TLR7 Expression Aggravates Invasive Pulmonary Aspergillosis by Suppressing Anti-Aspergillus Immunity of Macrophages.

Toll-like receptors (TLRs) play a critical role in early immune recognition of Aspergillus, which can regulate host defense during invasive pulmonary Aspergillosis (IPA). However, the role of TLR7 in the pathogenesis of IPA remains unknown. In this study, an in vivo model of IPA was established to investigate the contribution of TLR7 to host anti-Aspergillus immunity upon invasive pulmonary Aspergillus fumigatus infection. The effects of TLR7 on phagocytosis and killing capacities of A. fumigatus by macrophages and neutrophils were investigated in vitro We found that TLR7 knockout mice exhibited lower lung inflammatory response and tissue injury, higher fungal clearance, and greater survival in an in vivo model of IPA compared with wild-type mice. TLR7 activation by R837 ligand led to wild-type mice being more susceptible to invasive pulmonary Aspergillus fumigatus infection. Macrophages, but not neutrophils, were required for the protection against IPA observed in TLR7 knockout mice. Mechanistically, TLR7 impaired phagocytosis and killing of A. fumigatus by macrophages but not neutrophils. Together, these data identify TLR7 as an important negative regulator of anti-Aspergillus innate immunity in IPA, and we propose that targeting TLR7 will be beneficial in the treatment of IPA.

The Metabolic Cytokine Adiponectin Inhibits Inflammatory Lung Pathology in Invasive Aspergillosis.

Systemic immunity and metabolism are coregulated by soluble factors, including the insulin-regulating adipose tissue cytokine adiponectin. How these factors impact detrimental inflammatory responses during fungal infection remains unknown. In this study, we observed that mortality, fungal burden, and tissue histopathology were increased in adiponectin-deficient mice in a neutropenic model of invasive aspergillosis. Lung RNA sequencing, quantitative RT-PCR, and subsequent pathway analysis demonstrated activation of inflammatory cytokine pathways with upstream regulation by IL-1 and TNF in adiponectin-deficient mice with decreased/inhibited anti-inflammatory genes/pathways, suggesting broad cytokine-mediated pathology along with ineffective fungal clearance. Quantitative RT-PCR analysis confirmed increased transcription of IL-1a, IL-6, IL-12b, IL-17A/F, and TNF in adiponectin-deficient mice at early time points postinfection, with a specific increase in intracellular TNF in alveolar macrophages. Although eosinophil recruitment and activation were increased in adiponectin-deficient mice, mortality was delayed, but not decreased, in mice deficient in both adiponectin and eosinophils. Interestingly, neutrophil depletion was required for increased inflammation in adiponectin-deficient mice in response to swollen/fixed conidia, suggesting that immune suppression enhances detrimental inflammation, whereas invasive fungal growth is dispensable. Our results suggest that adiponectin inhibits excessive lung inflammation in invasive aspergillosis. Our study has therefore identified the adiponectin pathway as a potential source for novel therapeutics in immune-compromised patients with detrimental immunity to invasive fungal infection.

Therapeutic effects of pentoxifylline on invasive pulmonary aspergillosis in immunosuppressed mice.

BACKGROUND: The most common and severe infection of Aspergillus fumigatus is invasive pulmonary aspergillosis (IPA), which is usually seen in immunocompromised patients. Neutropenia is the primary risk factor implicated in IPA; however, IPA also occurs in patients without neutropenia, namely, those who are immunosuppressed owing to long-term corticosteroid use. With IPA-associated mortality as high as 51-79%, novel and effective treatment strategies are urgently needed. Pentoxifylline (PTX) has been shown to competitively inhibit the family 18 chitinases in fungi, which may be an new antifungal therapy. Hence, the aim of our study was to compare neutropenic and non-neutropenic IPA mouse models, and to evaluate the effect of PTX on IPA in immunosuppressed mice. METHODS: C57BL/6J mice were pre-treated with cyclophosphamide and hydrocortisone. Neutropenic model IPA mice (CTX-IPA) and non-neutropenic IPA mice (HC-IPA) were established by intranasal administration of Aspergillus fumigatus spore suspension. A subset of each group was injected with PTX post-infection. Among these groups, we compared overall survival, pulmonary fungal burden, lung hispathology, and myeloperoxidase (MPO), interleukin 8 (IL-8), and mammalian chitinase concentration in the bronchoalveolar lavage fluid (BALF). RESULTS: The survival rate of the HC-IPA group was higher than that of the CTX-IPA group, and pulmonary fungal burden was also lower (p < 0.05). The CTX-IPA group showed infiltration of alveolae and blood vessels by numerous hyphae of A. fumigatus. The HC-IPA group exhibited destruction of bronchi, expansion of alveolar septa, increased macrophages aggregation, significant neutrophil infiltration and a few hyphae in peribronchial areas. After PTX treatment, improvement was observed in survival duration and pulmonary fungal burden in HC-IPA mice. MPO and IL-8 levels were lower in the HC-IPA + PTX group compared to the corresponding levels in the HC-IP group. Chitotriosidase (CHIT1) and Chitinase 3-like 1 (CHI3L1) expression in the HC-IPA group was decreased after PTX treatment (p < 0.05). CONCLUSION: PTX was found to exert a therapeutic effect in a non-neutropenic mouse model of IPA, which may lead to the development of novel strategies for IPA treatment.

Protective Activity of Programmed Cell Death Protein 1 Blockade and Synergy With Caspofungin in a Murine Invasive Pulmonary Aspergillosis Model.

Pharmacological immune checkpoint blockade has revolutionized oncological therapies, and its remarkable success has sparked interest in expanding checkpoint inhibitor therapy in infectious diseases. Herein, we evaluated the efficacy of programmed cell death protein 1 (PD-1) blockade in a murine invasive pulmonary aspergillosis model. We found that, compared with isotype-treated infected control mice, anti-PD-1-treated mice had improved survival, reduced fungal burden, increased lung concentrations of proinflammatory cytokines and neutrophil-attracting chemokines, and enhanced pulmonary leukocyte accumulation. Furthermore, combined treatment with anti-PD-1 and caspofungin resulted in a significant survival benefit compared with caspofungin or anti-PD-1 therapy alone, indicating a synergistic effect between PD-1 inhibitors and immunomodulatory antifungal agents.

Galactomannan Is a Biomarker of Fosmanogepix (APX001) Efficacy in Treating Experimental Invasive Pulmonary Aspergillosis.

Galactomannan (GM) detection in biological samples has been shown to predict therapeutic response by azoles and polyenes. In a murine invasive pulmonary aspergillosis model, fosmanogepix or posaconazole treatment resulted in an ∼6- to 7-log reduction in conidial equivalents (CE)/g lung tissue after 96 h versus placebo. Changes in GM levels in BAL fluid and serum mirrored reductions in lung CE, with significant decreases seen after 96 h or 72 h for fosmanogepix or posaconazole, respectively (P < 0.02).

Chitinase Induction Prior to Caspofungin Treatment of Experimental Invasive Aspergillosis in Neutropenic Rats Does Not Enhance Survival.

Host chitinases, chitotriosidase and acidic mammalian chitinase (AMCase), improved the antifungal activity of caspofungin (CAS) against Aspergillus fumigatus in vitro These chitinases are not constitutively expressed in the lung. Here, we investigated whether chitosan derivatives were able to induce chitinase activity in the lungs of neutropenic rats and, if so, whether these chitinases were able to prolong survival of rats with invasive pulmonary aspergillosis (IPA) or of rats with IPA and treated with CAS. An oligosaccharide-lactate chitosan (OLC) derivative was instilled in the left lung of neutropenic rats to induce chitotriosidase and AMCase activities. Rats instilled with OLC or with phosphate-buffered saline (PBS) were subsequently infected with A. fumigatus and then treated with suboptimal doses of CAS. Survival, histopathology, and galactomannan indexes were determined. Instillation of OLC resulted in chitotriosidase and AMCase activities. However, instillation of OLC did not prolong rat survival when rats were subsequently challenged with A. fumigatus In 5 of 7 rats instilled with OLC, the fungal foci in the lungs were smaller than those in rats instilled with PBS. Instillation of OLC did not significantly enhance the survival of neutropenic rats challenged with A. fumigatus and treated with a suboptimal dosage of CAS. Chitotriosidase and AMCase activities can be induced with OLC, but the presence of active chitinases in the lung did not prevent the development of IPA or significantly enhance the therapeutic outcome of CAS treatment.

The evolving landscape of new diagnostic tests for invasive aspergillosis in hematology patients: strengths and weaknesses.

PURPOSE OF REVIEW: The diagnosis of invasive aspergillosis in hematologic patients is a complex composite of clinical preconditions and features, imaging findings, biomarker combinations from appropriate clinical samples and microbiological and/or histological findings. RECENT FINDINGS: Recent developments in the evolving landscape of diagnostic tests for invasive aspergillosis in adult hematology patients are highlighted. SUMMARY: Novel approaches and tools are currently under development. Focusing optimized diagnostic performance, in particular the combination of biomarkers from appropriate clinical samples, improved diagnostic performance distinctly.

[The expression of triggering receptor expressed on myeloid cells receptor-1 in Aspergillus infected mice].

Objective: To investigate the expression of triggering receptor expressed on myeloid cells receptor-1 (TREM-1) in plasma and bronchoalveolar lavage fluid (BALF) and its correlation with Galactomannan, IFNγ, IL-6 and IL-10 in Aspergillus infected mice. Methods: Cyclophosphamide(CTX) was intraperitoneally injected and fumigatus spore suspension was inhaled by nose to establish the immunocompromised invasive pulmonary aspergillosis(IPA) mouse model.Healthy controls, immunocompromised only and IPA only groups were also established. Each group had 6 mice. After inoculation, mice were sacrificed. Lung tissue specimens, BALF, and plasma samples were collected. Plasma and BALF soluble TREM-1 (sTREM-1), Galactomannan, IFNγ, IL-6, and IL-10 were detected by ELISA. Results: Positive Aspergillus fumigatus was found by tissue culture in the lung. Infiltration of inflammatory cells, blood congestion and interstitial lung tissue injury were observed in histological sections of both IPA and immunocompromised IPA mice. Compared to IPA group [(453.78±74.18) ng/L, P<0.001; (10.21±1.46) ng/L, P<0.001] and control group [(245.16±65.85) ng/L, P<0.001; (6.60±3.74) ng/L, P<0.001], the plasma and BALF sTREM-1 significantly increased in immunocompromised IPA group [(1 537.64±359.52) ng/L; (20.12±2.72) ng/L]. Compared to control group, both the BALF sTREM-1 in IPA group (P=0.041) and the plasma and BALF Galactomannan, IFNγ, IL-6, and IL-10 levels in IPA and immunocompromised IPA groups were significantly higher (P<0.01). Pearson correlation analysis showed that plasma and BALF sTREM-1 were significantly correlated with Galactomannan (r=0.83, P<0.001; r=0.82, P<0.001), IFNγ (r=0.79, P<0.001; r=0.61, P<0.01), IL-6 (r=0.81, P<0.001; r=0.66, P<0.01), and IL-10 (r=0.70, P=0.001; r=0.54, P=0.02). Conclusions: Plasma and BALF sTREM-1 appears highly expressed in Aspergillus infected mice. sTREM-1 in mice plasma and BALF is closely correlated with Galactomannan, IFNγ, IL-6, and IL-10 levels, which suggests that sTREM-1 has great diagnostic value during invasive fungal infection.

The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence.

Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize the A. fumigatus protein phosphatase 2A SitA, the Saccharomyces cerevisiae Sit4p homologue. The sitA gene is not an essential gene, and we were able to construct an A. fumigatus null mutant. The ΔsitA strain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ΔsitA strain is more sensitive to several metals and ions, such as MnCl2, CaCl2, and LiCl, but it is more resistant to ZnSO4. The ΔsitA strain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-α) response in mouse macrophages. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway.

[The expression of triggering receptor expresses on myeloid cells receptor-1, T cell-specific transcription factor, and eomesodermin in Aspergillus infected immunosuppressed rats].

OBJECTIVE: To investigate the function of triggering receptor expresses on myeloid cells receptor-1 (TREM-1) in lymphocyte differentiation and regulation of Aspergillus infected immunosuppressed rats. METHODS: Cyclophosphamide (CTX) was intraperitoneally injected and Fumigatus spore suspension was inhaled by percutaneous tracheostomy to establish the immunosuppressive invasive pulmonary aspergillosis (IPA) rat model. After 24 h, 48 h, 72 h and 96 h inoculation, rats were sacrificed. Lung tissue specimens, bronchoalveolar lavage fluid (BALF), and plasma samples were collected. Plasma and BALF sTREM-1, plasma T cell-specific transcription factor (T-box expressed in T cells, T-bet) and eomesodermin(Eomes) were detected by ELISA. Biopsy specimens of lung tissue were used for periodic acid-schiff (PAS) staining and culture. RESULTS: The mortality rate of immunosuppressed rats after Aspergillus inhalation for 96 h was as high as 54.4%. Biopsy of lung tissue suggested acute inflammatory cell infiltration, interstitial lung congestion, alveolar structural damage, and visible Aspergillus hyphae in alveoli. Compared with normal control group[(110.50 ± 7.70) ng/L], plasma sTREM-1 in study groups were significantly increased[IPA: (146.77 ± 10.41) ng/L; CXT+ IPA at 24 h: (226.00 ± 11.88) ng/L; CTX+ IPA at 48 h: (200.77 ± 10.63) ng/L; P<0.05], so were T-bet levels[IPA: (561.17 ± 7.23) µg/L; CXT+ IPA at 24 h: (647.00 ± 33.03) µg/L; CTX+ IPA at 48 h: (619.23 ± 87.44) µg/L; control group: (340.03 ± 26.32) µg/L; respectively, P < 0.05]. However, plasma Eomes levels in IPA group, CTX+ IPA at 24 h and 48 h were significantly lower compared with that in normal controls[IPA: (7.96 ± 0.65) ng/L; CXT+ IPA at 24 h: (3.97 ± 0.35) ng/L; CTX+ IPA at 48 h: (4.00 ± 0.74) ng/L; control group: (8.38 ± 0.51) ng/L; respectively, P < 0.001]. Compared with those in CTX+ IPA vaccination after 24 h and 48 h, plasma sTREM-1 [(106.67 ± 7.64) ng/L; (133.27 ± 32.79) ng/L] and T-bet [(299.64 ± 63.07) µg/L; (398.02 ± 109.22) µg/L] in CTX+ IPA at 72 h and 96 h inoculation were significantly lower (P < 0.001). While Eomes [(8.38 ± 0.54) ng/L; (8.40 ± 0.70) ng/L]raised significantly higher (P < 0.001). Compared with the control group, sTREM-1 levels in BALF of IPA+ CTX 24 h, 48 h, 72 h, and 96 h groups were consistently high (P < 0.05). Pearson correlation analysis showed that sTREM-1 and T-bet had a significant positive correlation (r = 0.91, P < 0.001), yet Eomes was negatively correlated with them (r = -0.788, P < 0.001). CONCLUSIONS: sTREM-1 in rat plasma and BALF appears highly expressed in immune compromised Aspergillus infected rat model. Plasma sTREM-1 is closely correlated with T-bet and Eomes levels, which suggests that TREM-1 may be involved in lymphocytic regulation and differentiation during fungal infection.

[The diagnostic performance of galactomannan detection for invasive pulmonary aspergillosis in non-neutropenic 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 pospective study was performed for 1 356 non-neutropenic hosts admitted to the Department of Pulmonary and Critical Care Medicine of the First Affiliated Hospital of Wenzhou Medical University from September 2014 to October 2015. Serum GM test was performed for all, and BALF GM test for a proportion of the patients. The patients were divided into an IPA group and a non-IPA group. SPSS 20.0 was adopted for statistical analysis. Results: A total of 1 361 cases were enrolled, aging 18-96 years, with an average age of (64±15) years. There were 879 male and 477 female patients. Thirty-nine cases were diagnosed as IPA, accounting for 2.9%. For serum GM test, the sensitivity, specificity, PPV and NPV were 43.6%(17/39), 94.1%(1 239/1 317), 17.9%(17/95)and 98.3%(1 239/1 261)respectively. Ninety-six cases received serum and BALF GM tests at the same time. If the cut-off value of BALF GM test was 0.8, the sensitivity, specificity, PPV and NPV were 86.7%(13/15), 60.5%(49/81), 28.9%(13/45), 96.1%(49/51)respectively, but if the value was 1.0, the sensitivity, specificity, PPV and NPV were 86.7%(13/15), 74.1%(60/81), 38.2%(13/34), 96.8%(60/62)respectively. The ROC curve area of BALF GM, serum GM and the combined serum and BALF GM was 0.87, 0.75 and 0.90, respectively. Conclusions: The sensitivity of serum GM test in non-neutropenic hosts was low, but it had a high negative predictive value.The best BALF GM cut-off value was 1.0. The combined serum and BALF GM tests improved the diagnostic performance.

Intrapulmonary posaconazole penetration at the infection site in an immunosuppressed murine model of invasive pulmonary aspergillosis receiving oral prophylactic regimens.

Adequate penetration to the infection/colonization site is crucial to attain optimal efficacy of posaconazole against Aspergillus fumigatus diseases. We evaluated posaconazole exposure in pulmonary epithelial lining fluid (ELF) in a murine model of invasive pulmonary aspergillosis. The posaconazole exposure (area under the plasma concentration-time curve from time zero to 24 h postinfusion [AUC0-24]) in ELF was 20% to 31% of that in plasma for total drug after the third dose, and the relationship between plasma and ELF exposure was linear (r(2) = 0.97, P = 0.016).

Efficacy and safety of short- and long-term treatment of itraconazole on chronic necrotizing pulmonary aspergillosis in multicenter study.

In the respiratory field, chronic pulmonary aspergillosis, such as chronic necrotizing pulmonary aspergillosis (CNPA) or aspergilloma, is important. We examined the efficacy and safety of short- and long-term itraconazole (ITCZ) administration, involving a switch from injection to an oral preparation, in patients with CNPA. In all hospitals participating in this study, the protocol was approved by the ethics review board. This study started after UMIN registration (UMIN000001727). Subjects enrolled in this study were patients who were clinically or definitively diagnosed with CNPA in the respiratory field, according to the diagnostic criteria of the Japanese "Guidelines for management of deep-seated mycosis 2007," in 16 hospitals that participated in this study between May 2008 and March 2011. Treatment was started with ITCZ injection. Subsequently, the agent was switched to an oral preparation. Efficacy was evaluated with major items (clinical symptoms, fever, imaging findings) and minor items (nutritional status, inflammatory markers). Twenty-nine patients were enrolled; safety was evaluated in 24 and efficacy in 23. Of the 23 patients, 10 (43.5 %) responded. With respect to the administration period, the response rates in 8 patients treated for a short period and 15 treated for a long period were 25.0 % and 53.3 %, respectively. Trough blood concentration of ITCZ reached a level at which ITCZ may be effective for aspergillosis at 3 days after the start of ITCZ injection therapy. After changing to high-dose capsules, its level was also maintained. Adverse events such as liver dysfunction and heart failure were observed in 9 of the 24 patients. Furthermore, 6 patients died. However, there was no relationship between these events and ITCZ. Step-down therapy from ITCZ injection to oral administration may be a useful treatment option in CNPA patients requiring long-term treatment.

Detection of Low Oxygen Microenvironments in a Murine Model of Invasive Pulmonary Aspergillosis Using Pimonidazole.

Infection tissue microenvironments are dynamic, complex, and play a critical role in host-microbe interaction outcomes. A crucial parameter of the infection site microenvironment is oxygen. Both host and microbial cell physiology is significantly impacted by the availability of oxygen. When oxygen tensions drop to levels that do not meet the metabolic demands of the cell, a hypoxia response ensues. In numerous host-microbe studies, it has now been observed that the host and microbial hypoxia response plays a critical role in disease outcomes. However, in most pathosystems, spatial and temporal oxygen dynamics throughout the infection remain ill defined. Here, we detail a protocol for detecting low oxygen environments in tissue in a murine model of invasive pulmonary aspergillosis. The protocol utilizes mice immune compromised with a high dose of steroid and challenged via the aerosol route with conidia of the major human fungal pathogen Aspergillus fumigatus. Qualitative analysis of oxygen levels at the site of infection in the murine lung is accomplished with pimonidazole-mediated adduct detection via immunohistochemistry. The protocol is adaptable to other host-microbe interaction models.

FIBCD1 Deficiency Decreases Disease Severity in a Murine Model of Invasive Pulmonary Aspergillosis.

Aspergillus fumigatus is a ubiquitous mold associated with the development of pulmonary diseases that include invasive pulmonary aspergillosis (IPA), an often fatal opportunistic infection. FIBCD1 is a transmembrane endocytic membrane receptor widely expressed on human epithelium. Although FIBCD1 was previously shown to bind chitin, modulate fungal colonization of the gut, and inhibit intestinal inflammation, the role of FIBCD1 in the context of lung fungal infection remains unknown. In this study, we observed that mortality, fungal burden, and tissue histopathology were decreased in the absence of FIBCD1 in murine IPA. Quantitative RT-PCR analyses demonstrated decreased inflammatory cytokines in the lungs of neutrophil-depleted FIBCD1-/- mice with IPA, when compared with wild-type controls. In contrast, inflammatory cytokines were increased in immune-competent FIBCD1-/- mice after fungal aspiration, suggesting that the presence of neutrophils is associated with cytokine modulation. In contrast to the clear IPA phenotype, FIBCD1-/- mice with systemic infection or bleomycin-induced lung injury exhibited similar morbidity and mortality when compared with their wild-type counterparts. Thus, our study identifies a detrimental role of FIBCD1 in IPA.

[Synthetic Study of Polycyclic Natural Products Based on Development of New Strategy].

On the occasion of receiving the Pharmaceutical Society of Japan Award 2020, I explained our research activities on the total syntheses of polycyclic alkaloids as an invited review. The structure of lundurine B, which has an unstable cyclopropane fused indoline skeleton, was proved firstly by the total synthesis. I also describe the total syntheses of optically active lundurine B and rapidilectine B utilizing asymmetric desymmetrization of the spiro intermediate. We developed an intramolecular bond formation reaction between the 2-position of the furan ring to the iminium cation (furane-iminium cation cyclization) to synthesize manzamine alkaloids. The reaction was applied to the total synthesis of the core skeleton of nakadomarin A and ircinal A. A ring-closing metathesis reaction effectively applied to the synthesis of medium and large heterocyclic rings containing the cis double bond found in the structures of nakadomarin A and ircinal A. The total synthesis of schizocommunin, a metabolite of Schizophyllum commune isolated from a patient with human allergenic bronchopulmonary mycosis, was accomplished. We could correct the error in the proposed structure by total synthesis of the natural product. A part of the mechanism of cytotoxicity expression was clarified using newly synthesized shizocommunin.

The initial 96 hours of invasive pulmonary aspergillosis: histopathology, comparative kinetics of galactomannan and (1->3) ß-d-glucan and consequences of delayed antifungal therapy.

Acute invasive pulmonary aspergillosis is a rapidly progressive and frequently lethal infection. Relatively little is known about early events in the pathogenesis and relationship between the cell wall biomarkers galactomannan and (1→3)-ß-d-glucan. The consequences of delayed antifungal therapy are also poorly defined. A persistently neutropenic rabbit model of invasive pulmonary aspergillosis was used to describe the histopathology of early invasive pulmonary aspergillosis and the kinetics of galactomannan and (1→3)-ß-d-glucan. The time course of both molecules was mathematically modeled by using a population methodology, and Monte Carlo simulations were performed. The effect of progressive delay in the administration of amphotericin B deoxycholate 1 mg/kg at 24, 48, 72, and 96 h postinoculation on fungal burden, lung weight, pulmonary infarct score, and survival was determined. Histopathology showed phagocytosis of conidia by pulmonary alveolar macrophages at 4 h postinoculation. At 12 to 24 h, there was a progressive focal inflammatory response with conidial germination and hyphal extension. Subsequently, hyphae invaded into the contiguous lung. Galactomannan and (1→3)-ß-d-glucan had similar trajectories, and both exhibited considerable interindividual variability, which was reflected in Monte Carlo simulations. Concentrations of both molecules began to rise <24 h postinoculation before pulmonary hemorrhagic infarction was present. Delays of 72 and 96 h in the administration of amphotericin B resulted in fungal burdens and lung weights that were indistinguishable from those of controls, respectively. Galactomannan and (1→3)-ß-d-glucan have similar kinetics and are comparable biomarkers of early invasive pulmonary aspergillosis. Antifungal treatment at ≥48 h postinoculation is associated with suboptimal therapeutic outcomes.

In vivo bioluminescence imaging and histopathopathologic analysis reveal distinct roles for resident and recruited immune effector cells in defense against invasive aspergillosis.

BACKGROUND: Invasive aspergillosis (IA) is a major cause of infectious morbidity and mortality in immune compromised patients. Studies on the pathogenesis of IA have been limited by the difficulty to monitor disease progression in real-time. For real-time monitoring of the infection, we recently engineered a bioluminescent A. fumigatus strain. RESULTS: In this study, we demonstrate that bioluminescence imaging can track the progression of IA at different anatomic locations in a murine model of disease that recapitulates the natural route of infection. To define the temporal and functional requirements of distinct innate immune cellular subsets in host defense against respiratory A. fumigatus infection, we examined the development and progression of IA using bioluminescence imaging and histopathologic analysis in mice with four different types of pharmacologic or numeric defects in innate immune function that target resident and recruited phagocyte subsets. While bioluminescence imaging can track the progression and location of invasive disease in vivo, signals can be attenuated by severe inflammation and associated tissue hypoxia. However, especially under non-inflammatory conditions, such as cyclophosphamide treatment, an increasing bioluminescence signal reflects the increasing biomass of alive fungal cells. CONCLUSIONS: Imaging studies allowed an in vivo correlation between the onset, peak, and kinetics of hyphal tissue invasion from the lung under conditions of functional or numeric inactivation of phagocytes and sheds light on the germination speed of conidia under the different immunosuppression regimens. Conditions of high inflammation -either mediated by neutrophil influx under corticosteroid treatment or by monocytes recruited during antibody-mediated depletion of neutrophils- were associated with rapid conidial germination and caused an early rise in bioluminescence post-infection. In contrast, 80% alveolar macrophage depletion failed to trigger a bioluminescent signal, consistent with the notion that neutrophil recruitment is essential for early host defense, while alveolar macrophage depletion can be functionally compensated.

Diagnostic Accuracy of Bronchoalveolar Lavage Fluid Galactomannan for Invasive Aspergillosis.

BACKGROUND: The pathogenesis of invasive aspergillosis (IA) is still unknown, but its progression is rapid and mortality rate remains high. Bronchoalveolar lavage fluid (BALF) galactomannan (GM) analysis has been used to diagnose IA. This study is aimed at making an accurate estimate of the whole accuracy of BALF-GM in diagnosing IA. METHODS: After a systematic review of the study, a bivariate meta-analysis was used to summarize the specificity (SPE), the sensitivity (SEN), the positive likelihood ratios (PLR), and the negative likelihood ratios (NLR) of BALF-GM in diagnosing IA. The overall test performance was summarized using a layered summary receiver operating characteristic (SROC) curve. Subgroup analysis was performed to explore the heterogeneity between studies. RESULTS: A total of 65 studies that are in line with the inclusion criteria were included. The summary estimates of BALF-GM analysis are divided into four categories. The first is the proven+probable vs. possible+no IA, with an SPE, 0.87 (95% CI, 0.85-0.98); SEN, 0.81 (95% CI, 0.76-0.84); PLR, 9.78 (5.78-16.56); and NLR, 0.20 (0.14-0.29). The AUC was 0.94. The BALF-GM test for proven+probable vs. no IA showed SPE, 0.88 (95% CI, 0.87-0.90); SEN, 0.82 (95% CI, 0.78-0.85); PLR, 6.56 (4.93-8.75); and NLR, 0.24 (0.17-0.33). The AUC was 0.93. The BALF-GM test for proven+probable+possible vs. no IA showed SPE, 0.82 (95% CI, 0.79-0.95); SEN, 0.59 (95% CI, 0.55-0.63); PLR, 3.60 (2.07-6.25); and NLR, 0.31 (0.15-0.61). The AUC was 0.86. The analyses for others showed SPE, 0.85 (95% CI, 0.83-0.87); SEN, 0.89 (95% CI, 0.86-0.91); PLR, 6.91 (4.67-10.22); and NLR, 0.18 (0.13-0.26). The AUC was 0.94. CONCLUSIONS: The findings of this BALF-GM test resulted in some impact on the diagnosis of IA. The BALF-GM assay is considered a method for diagnosing IA with high SEN and SPE. However, the patients' underlying diseases may affect the accuracy of diagnosis. When the cutoff is greater than 1, the sensitivity will be higher.