Progress in diagnosis and treatment of invasive aspergillosis after liver transplantation

Invasive aspergillosis is an opportunistic fungal infection disease,and the risk factors of Invasive aspergillosis after liver transplantation are increasing,which seriously affects the quality of life of patients.Invasive Aspergillus has no specific clinical manifestations and occurs most frequently in the lungs.The diagnostic methods for invasive aspergillosis are continuously updated,including serological tests,polymerase chain reaction(PCR),next-generation sequencing,Matrix-assisted laser desorption ionization time-of-flight mass spectrometry,Aspergillus GM lateral flow test,and some new markers under study have made the diagnosis of invasive aspergillosis infection more definitive.Voriconazole is the drug of choice for the treatment and prevention of invasive aspergillosis,and immunotherapy may become an adjuvant therapy or monotherapy for invasive aspergillosis with the emergence of more and more resistant strains.This article summarizes the progress in the diagnosis and treatment of invasive aspergillosis after liver transplantation,in order to provide a reference for clinical practice.

Existing tests vs. novel non-invasive assays for detection of invasive aspergillosis in patients with respiratory diseases

Background: Although existing mycological tests (bronchoalveolar lavage [BAL] galactomannan [GM], serum GM, serum (1,3)-β-D-glucan [BDG], and fungal culture) are widely used for diagnosing invasive pulmonary aspergillosis (IPA) in non-hematological patients with respiratory diseases, their clinical utility in this large population is actually unclear. We aimed to resolve this clinical uncertainty by evaluating the diagnostic accuracy and utility of existing tests and explore the efficacy of novel sputum-basedAspergillus assays.Methods: Existing tests were assessed in a prospective and consecutive cohort of patients with respiratory diseases in West China Hospital between 2016 and 2019 while novel sputum assays (especially sputum GM andAspergillus-specific lateral-flow device [LFD]) in a case-controlled subcohort. IPA was defined according to the modified European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Sensitivity and specificity were computed for each test and receiver operating characteristic (ROC) curve analysis was performed.Results: The entire cohort included 3530 admissions (proven/probable IPA=66, no IPA=3464) and the subcohort included 127 admissions (proven/probable IPA=38, no IPA=89). Sensitivity of BAL GM (≥1.0 optical density index [ODI]: 86% [24/28]) was substantially higher than that of serum GM (≥0.5 ODI: 38% [39/102]) (χ^(2)=19.83,P<0.001), serum BDG (≥70 pg/mL: 33% [31/95]) (χ^(2)=24.65,P<0.001), and fungal culture (33% [84/253]) (χ^(2)=29.38,P<0.001). Specificity varied between BAL GM (≥1.0 ODI: 94% [377/402]), serum GM (≥0.5 ODI: 95% [2130/2248]), BDG (89% [1878/2106]), and culture (98% [4936/5055]). Sputum GM (≥2.0 ODI) had similar sensitivity (84% [32/38]) (Fisher’s exactP=1.000) to and slightly lower specificity (87% [77/89]) (χ^(2)=5.52,P=0.019) than BAL GM (≥1.0 ODI). Area under the ROC curve values were comparable between sputum GM (0.883 [0.812-0.953]) and BAL GM (0.901 [0.824-0.977]) (P=0.734). Sputum LFD had similar specificity (91% [81/89]) (χ^(2)=0.89,P=0.345) to and lower sensitivity (63% [24/38]) (χ^(2)=4.14,P=0.042) than BAL GM (≥1.0 ODI), but significantly higher sensitivity than serum GM (≥0.5 ODI) (χ^(2)=6.95,P=0.008), BDG (χ^(2)=10.43,P=0.001), and fungal culture (χ^(2)=12.70,P<0.001).Conclusions: Serum GM, serum BDG, and fungal culture lack sufficient sensitivity for diagnosing IPA in respiratory patients. Sputum GM and LFD assays hold promise as rapid, sensitive, and non-invasive alternatives to the BAL GM test.

Therapeutic potential of Calotropis gigantea extract against invasive pulmonary aspergillosis:In vitro and in vivo study

Objective:To characterize the antifungal activity of methanolic leaf extract of Calotropis gigantea alone or in combination with amphotericin B against invasive pulmonary aspergillosis in mice.Methods:GC/MS was used for analysis of active constituents of Calotropis gigantea extract.Spore germination assay and broth micro-dilution method were used to determine antifungal potential of Calotropis gigantea/amphotericin B against Aspergillus fumigatus.Neutropenic mice were randomly assigned into 5 groups:group 1 was neutropenic(control);group 2 was infected with Aspergillus fumigatus;group 3 was infected with Aspergillus fumigatus,and treated with Calotropis gigantea extract;group 4 was infected with Aspergillus fumigatus and treated with amphotericin B;group 5 was infected with Aspergillus fumigatus and treated with both Calotropis gigantea extract and amphotericin B.Fresh lung tissues were histopathologically examined.Fungal burden and gliotoxin concentration were evaluated in lung tissues.Catalase,superoxide dismutase,and malondialdehyde content were determined in lung tissues.Myeloperoxidase,tumor necrosis factor-alpha,interleukin-1,and interleukin-17 were also estimated by the sandwich enzyme-linked immuno-sorbent assay.Results:Calotropis gigantea/amphotericin B had a minimum inhibitory concentration and minimum fungicidal concentration of 80 and 160μg/mL,respectively,for Aspergillus fumigatus.Additionally,Calotropis gigantea/amphotericin B significantly reduced lung fungal burden by 72.95%and inhibited production of gliotoxin in lung tissues from 6320 to 1350μg/g lung.Calotropis gigantea/amphotericin B reduced the oxidative stress of the lung via elevating the activity of antioxidant enzymes and decreasing the levels of lipid peroxidation.Myeloperoxidase activity and the production of pro-inflammatory cytokines were also significantly reduced.Scanning electron microscopy revealed deteriorations in the hyphae ultrastructure in Calotropis gigantea/amphotericin B treated Aspergillus fumigatus and leak of cellular components after damage of the cell wall.In vivo study revealed the suppression of lung tissue damage in mice of invasive pulmonary aspergillosis,which was improved with Calotropis gigantea/amphotericin B compared to the control group.Conclusions:Calotropis gigantea/amphotericin B is a promising treatment to reduce lung fungal burden and to improve the drugs’therapeutic effect against invasive pulmonary aspergillosis.

A Case of Invasive Pulmonary Aspergillosis Resulted from the Treatment of Chronic Eczema

This was an advanced male(87-year-old)with refractory chronic eczema for over 40 years,based on his allergic constitution,accompanied with chronic kidney disease due to primary hypertension(CKD,phase 3).It was so difficult to tolerate the severe itching that the glucocorticoids(GC)had to be applied to it,but some new-onset respiratory symptoms,such as cough,dyspnea after exertion etc.,occurred to this patient.Some classical IPA images were found on his pulmonary CT scanning,which were further comfirmed by the positive findings of GM-test,and then a final diagno­sis of IPA was accordingly established.Unfortunately,a persistent fever emerged after starting an antifungal therapy to the patient,and his IL-2 level was detected to be superhigh.As a response to allergic fever,GC was carefully given intravenously again to treat it,and it turned out to be totally improved since then;suggesting that systemic thinking(integrated with the other clinical evidences)is essential to diagnose IPA,and GC can also be used to improve its symptoms with the existence of antifungal therapy.

Significance of Aspergillus spp. isolation from lower respiratory tract samples for the diagnosis and prognosis of invasive pulmonary aspergillosis in chronic obstructive pulmonary disease

Background Chronic obstructive pulmonary diseases （COPD） is an emerging population at risk for invasive infection of Aspergillus. Isolation of Aspergillus from lower respiratory tract （LRT） samples is important for the diagnosis of invasive pulmonary aspergillosis （IPA）. The purpose of this study was to investigate the value of Aspergillus isolation from LRT samples for the diagnosis and prognosis of IPA in COPD population. Methods Clinical record with Aspergillus spp. isolation in COPD and immunocompromised patients was reviewed in a retrospective study. Patients were categorized and compared according to their severity of illness （admitted to general ward or ICU） and immunological function （COPD or immunocompromised）. Results Multivariate statistical analysis showed that, combined with Aspergillus spp. isolation, APACHE II scores 〉18, high cumulative doses of corticosteroids （〉350 mg prednisone or equivalent dose） and more than four kinds of broad-spectrum antibiotics received in hospital may be predictors of IPA in COPD （0R=9.076, P=0.001; 0R=4.073, P=-0.026; OR=4.448, P=-0.021, respectively）. The incidence of IPA, overall mortality, mortality of patients with IPA and mortality of patients with Aspergillus spp. colonization were higher in COPD patients in ICU than in general ward, but were similar between COPD and immunocompromised patients. Conclusions Aspergillus spp. isolation from LRT in COPD may be of similar importance as in immunocompromised patients, and may indicate an increased diagnosis possibility of IPA and worse prognosis when these patients received corticosteroids, antibiotics, and need to admit to ICU. Aspergillus spp. isolation from LRT samples combined with certain risk factors mav be useful in differentiating colonization from IPA and evaluating the prognosis of IPA in COPD patients.

Role of Triggering Receptor Expressed on Myeloid Cell-1 Expression in Mammalian Target of Rapamycin Modulation of CD8＋ T-cell Differentiation during the Immune Response to Invasive Pulmonary Aspergillosis

Background： Triggering receptor expressed on myeloid cell- 1 （TREM- 1） may play a vital role in mammalian target ofrapamycin （mTOR） modulation ofCD8＋ T-cell differentiation through the transcription factors T-box expressed in T-cells and eomesodermin during the immune response to invasive pulmonary aspergillosis （IPA）. This study aimed to investigate whether the roTOR signaling pathway modulates the proliferation and differentiation of CD8＋ T-cells during the immune response to I PA and the role TREM-1 plays in this process. Methods： Cyclophosphamide （CTX） was injected intraperitoneally, and Asl？e;gillus.[mnigams spore suspension was inoculated intranasally to establish the immunosuppressed IPA mouse model. After inoculation, rapamycin （2 mg-kg ·d -1） or interleukin （IL）-12 （5 μg/kg every other day） was given for 7 days. The number of CD8＋ effector memory T-cells （Tern）, expression of interferon （IFN）-y, roTOR, and ribosomal protein $6 kinase （S6K）, and the levels of IL-6, IL- 10, galactomannan （GM）, and soluble TREM- 1 （sTREM-I） were measured. Results： Viable A. fumigatus was cultured from the lung tissue of the inoculated mice. Histological examination indicated greater inflammation, hemorrhage, and lung tissue injury in both IPA and CTX ＋ IPA mice groups. The expression of mTOR and S6K was significantly increased in the CTX ＋ IPA ＋ I L- 12 group compared with the control, I PA （P = 0.01 ; P - 0.001 ）, and CTX ＋ 1PA （P = 0.034; P = 0.032） groups, but significantly decreased in the CTX ＋ IPA ＋ RAPA group （P 〈 0.001 ）. Compared with the CTX ＋ IPA group, the proportion of Tern, expression of IFN-y, and the level ofsTREM-I were significantly higher after IL-12 treatment （P = 0.024, P = 0.032, and P = 0.017, respectively）, and the opposite results were observed when the roTOR pathway was blocked by rapamycin （P 〈 0.001）. Compared with the CTX ＋ I PA and CTX ＋ I PA ＋ RAPA groups, IL-12 treatment increased IL-6 and downregulated IL- 10 as well as G M, which strengthened the immune response to the IPA infection. Conclusions： mTOR modulates CD8＋ T-cell differentiation during the immune response to IPA. TREM-1 may play a vital role in signal transduction between mTOR and the downstream immune response.

Initial computed tomography findings of invasive pulmonary aspergillosis in non-hematological patients

Background The computed tomography （CT） findings of invasive pulmonary aspergillosis （IPA） are unclear in non- hematological patients. The present study was a retrospective evaluation of CT images in non-hematological patients with IPA. Methods All adult patients who met the 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group （EORTC/MSG） criteria for proven or probable IPA were included during a 5-year study at our institutions. Initial CT findings in our cohort were retrospectively reviewed by two independent thoracic radiologists blinded to patient demographics and clinical outcomes. The presence, pattern, and distribution of abnormalities were recorded. Results Twenty-three non-hematological patients with pathologically confirmed IPA were included in our study. Areas of ground-glass opacities were present in 14 patients （61%）, which were bilateral in 10 patients and unilateral in four. This pattern mainly involved the middle and upper lung zones. Air-space consolidation was identified in 12 patients （52%）, and the areas were distributed along the bronchus or subpleura in most cases. Other findings, including five small nodules （22%）, three macronodules （13%）, and one halo sign （4%）, were less common. Conclusions CT findings of IPA in non-hematological patients frequently manifested as acute bronchopneumonia, and ground-glass opacities and air-space consolidations were the most common CT findings of IPA in these patients.

Prognostic value of serum galactomannan index in critically ill patients with chronic obstructive pulmonary disease at risk of invasive pulmonary aspergillosis

Background Critically ill chronic obstructive pulmonary disease （COPD） patients admitted to an intensive care unit （ICU） due to respiratory failure are at particularly high risk of Aspergillus infection.The serum galactomannan index （GMI） has proven to be one of the prognostic criteria for invasive pulmonary aspergillosis （IPA） in classical immunocompromised patients.However,the prognostic value of serum GMI in critically ill COPD patients needs evaluation.The purpose of this study is to investigate the prognostic value of serum GMI in patients with severe COPD.Methods In this single-center prospective cohort study,serum samples for GMI assay were collected twice a week from the first day of ICU admission to the day of the patients＇ discharge or death.Patients were divided into two groups according to their clinical outcome on the 28th day of their ICU admission.Univariate analysis and survival analysis were tested in these two groups.Results One hundred and fifty-three critically ill COPD patients were included and were divided into survival group （106 cases） and non-survival group （47 cases） according to their outcome.Univariate analysis showed that the highest GMI level during the first week after admission （GMI-high 1st week） was statistically different between the two groups.Independent prognostic factors for poor outcome in severe COPD patients were：GMI-high 1st week ＞0.5 （RR：4.04,95％ CI：2.17-7.51） combined with accumulative dosage of corticosteroids ＞216 mg before the RICU admission （RR：2.25,95％ CI：1.11-4.56） and clearance of creatinine （Ccr） ＜64.31 ml/min （RR：2.48,95％ CI：1.22-5.07）.Conclusions The positive GMI-high 1st week （＞0.5） combined with an accumulative dosage of corticosteroids ＞216 mg before the ICU admission and a low Ccr may predicate a poor outcome of critically ill COPD patients.

Altered CD8＋ T-cell counts as an early predictor of prognosis in critically ill immunocompromised patients with invasive pulmonary aspergillosis

Background The number of critically ill immunocompromised （CIIC） patients has increased dramatically in recent years,and they represent a high risk population for invasive pulmonary aspergillosis （IPA） infection.Host immunity should play a major role in determining the outcome and recovery of these patients.The purpose of this study was to evaluate the dynamic changes in host immune status and its potential influence on prognosis in CIIC patients with IPA.Methods We monitored the evolution of a number of key cellular and humoral parameters on days 1,3,and 10 （D1,D3 and D10） following ICU admission in sixty-two CIIC patients with microbiological evidence of IPA.We included immunoglobulins IgG,IgA and IgM,complement factors C3 and C4,and lymphocyte subgroups CD3＋,CD4＋,CD8＋,CD28＋CD4＋,and CD28＋CD8＋ T cells,CD19＋B cells,and CD3-CD16＋CD56＋ natural killer cells （NK）.Results The primary outcome was 28-day mortality.Thirty-eight （61.3％） patients died within the 28 days following ICU admission.Compared to patients who died,CD3＋,CD8＋,CD28＋CD8＋ T-cell counts on D1,D3,and D10,CD28＋CD4＋ T-cell counts on D3 and D10,and NK counts on D3 and D10 were significantly higher in survivors.Receiver operating characteristic （ROC） analysis of immune parameters predicting 28-day mortality revealed area under the curve （AUC） values of 0.82 （95％ CI 0.71-0.92）,0.94 （95％ CI 0.87-0.99）,and 0.94 （95％ CI 0.85-0.99） for CD8＋ T-cell counts for D1,D3,and D10 respectively,and 0.84 （95％ CI 0.75-0.94）,0.92 （95％ CI 0.85-0.99）,and 0.90 （95％ CI 0.79-0.99） for CD28＋CD8＋ T-cell counts for D1,D3,and D10 respectively.Kaplan-Meier survival analysis showed that CD8＋ T-cell counts ＜149.5×106 cells/L and CD28＋CD8＋ T-cell counts ＜75×106 cells/L at ICU admission were associated with lower survival probabilities in CIIC patients with IPA （both Log rank：P＜0.001）.Conclusions Low CD8＋ and CD28＋CD8＋ T-cell counts were associated with high mortality in CIIC patients with IPA.Early counts of CD8＋ and CD28＋CD8＋ T cells in CIIC patients with IPA may be valuable for predicting outcome.

Th17 cells are involved in mouse chronic obstructive pulmonary disease complicated with invasive pulmonary aspergillosis

Background:The incidence of chronic obstructive pulmonary disease(COPD)complicated with invasive pulmonary aspergillosis(IPA)has increased in the last two decades.The mechanism underpinning susceptibility to and high mortality of COPD complicated with IPA is unclear,and the role of T helper cells 17(Th17 cells)in the compound disease remains unknown.Therefore,this study aimed to assess the function of Th17 cells in COPD combined with IPA.Methods:COPD,IPA,and COPD+IPA mouse models were established in male wild type C57/BL6 mice.The amounts of Th17 cells and retinoic acid-related orphan receptorsγt(RORyt)were tested by flow cytometry.Then,serum interleukin(IL)-17 and IL-23.levels were detected by enzyme-linked immunosorbent assay(ELISA)in the control,COPD,IPA and COPD+IPA groups.In addition,COPD+IPA was induced in IL-17 knockout(KO)mice,for determining the role of Th17 cells in COPD+IPA.Results:Compared with the COPD group,the COPD+IPA group showed higher amounts of blood RORyt([35.09±16.12]%vs.[17.92±4.91]%,P=0.02)and serum IL-17(17.96±9.59 pg/mL vs.8.05±4.44 pg/mL,P=0.02),but blood([5.18±1.09]%vs.[4.15±0.87]%,P=0.28)and lung levels of Th17 cells(1.98±0.83]%vs.[2.03±0.98]%,P=0.91),lung levels of RORyt([9.58±6.93]%vs.[9.63±5.98]%,P=0.49)and serum IL-23(51.55±27.82 pg/mL us.68.70±15.20 pg/mL,P=0.15)showed no significant differences.Compared with the IPA group,the COPD+IPA group displayed lower amounts of blood([5.18±1.09]%vs.[9.21±3.56]%,P=0.01)and lung Th17 cells([1.98±0.83]%vs.[6.29±1.11]%,P=0.01)and serum IL-23(51.55±27.82 pg/mL vs.154.90±64.60 pg/mL,P=0.01)and IL-17(17.96±9.59 pg/mL uUs.39.81±2.37 pg/mL,P=0.02),while comparable blood([35.09±16.12]%Vs.[29.86±15.42]%,P=0.25)and lung levels of RORγt(9.58±6.93]%VUS,[15.10±2.95]%,P=0.18)were found in these two groups.Finally,Aspergillus load in IL-17 KO COPD+IPA mice was almost 2 times that of COPD+IPA mice(1,851,687.69±944,480.43"vs.892,958.10±686,808.80,t=2.32,P=0.02).Conclusion:These findings indicate that Th17 cells might be involved in the pathogenesis of COPD combined with IPA,with L-17 likely playing an antifungal role.

CYP2C19 Genotyping Plus Therapeutic Drug Monitoring Dependent Voriconazole Treatment for Invasive Pulmonary Aspergillosis in a Patient with Liver Failure

Invasive pulmonary aspergillosis(IPA)is a lethal infectious disease with high mortality in patients with liver failure.Early recognition of the risk factors prompting earlier diagnosis and treatment may improve the outcomes.Voriconazole is recommended as the first-line drug for IPA,but hepatotoxicity limits its use in the context of liver diseases.We report a case of a 63-year-old female who was admitted to the Third Affiliated Hospital of Hebei Medical University due to IPA after glucocorticoid therapy for liver failure.The polymorphism of cytochrome P450(CYP)isoenzymes showed CYP2C19∗1/∗2 genotype associated with intermediate metabolism of voriconazole.However,the patient developed side effects such as skin rash,vomiting,hyperbilirubinemia,and alteration of consciousness,even if she received half of the recommended dosage for voriconazole.Therapeutic drug monitoring(TDM)was applied to guide the dosage adjustment of voriconazole in this patient,and consequently,the patient presented a favorable outcome.In conclusion,genotyping screening plus TDM dependent individualized treatment of voriconazole may improve the survival of liver failure patient with IPA.

The Diagnostic performance of galactomannan detection for invasive pulmonary aspergillosis in non-nentropenic hosts

Objective To evaluate the diagnostic performance of galactomannan(GM)detection in serum and BALF for invasive pulmonary aspergillosis(IPA)in non-neutropenic hosts.Methods A prospective study was performed for 1 356 non-neutropenic hosts admitted to the Department of Pulmonary and Critical Care Medicine of

Application Value of Macrogene Ⅱ Sequencing in IPA Diagnosis and Treatment

Infectious diseases feature multiple pathogens,complicated and diverse clinical symptoms.Early and accurate detection of infectious pathogens sets the foundation for its targeted clinical treatment.With long detection cycles and low positive rates,traditional detection approaches such as Gram-stained smear microscopy and specific marker detection can barely meet the clinical needs for infectious disease detection.As a new gene detection technology,macrogeneⅡsequencing can enable higher detection efficiency for infectious diseases caused by pathogens such as fungi and bacteria,demonstrating certain application value.This paper describes invasive pulmonary aspergillosis(IPA),concepts concerning macrogeneⅡsequencing,as well as its application value for invasive pulmonary aspergillosis.