Targeted DPPC/DMPG surface-modified voriconazole lipid nanoparticles control invasive pulmonary aspergillosis in immunocompromised population: in-vitro and in-vivo assessment.

Invasive pulmonary aspergillosis (IPA) is the most devastating Aspergillus-related lung disease. Voriconazole (VRZ) is the first-line treatment against IPA. Despite availability in oral and parenteral dosage forms, risks of systemic toxicity dictate alternative pulmonary administration. Inspired by natural lung surfactants, dipalmitoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DPPC/DMPG) surface-modified lipid nanoparticles (LNPs) were scrutinized for pulmonary administration. DPPC/DMPG-VRZ-LNPs prepared using ultrasonication/thin film hydration were investigated for colloidal properties over 3-month shelf storage. They were stable with a slight change in entrapment efficiency. They provided a sustained VRZ release over 24 h, with a rapid initial release. In vitro aerosolization indicated higher percentages of VRZ deposited on stages corresponding to secondary bronchi and alveolar ducts. Moreover, intrapulmonary administration maintained high lung VRZ concentration (27 ± 1.14 µg/g) after 6 h. A preclinical study using a cyclophosphamide-induced neutropenic rat model demonstrated a 3-fold reduction in BALF-Galactomannan down to 0.515 ± 0.22 µg/L confirming DPPC/DMPG-VRZ-LNPs potential in hyphal growth inhibition. Histopathological examination of infected/nontreated lung sections exhibited dense fungal load inside alveoli and blood vessels indicating massive tissue and angio-invasiveness. Nevertheless, DPPC/DMPG-VRZ-LNPs-treated animals displayed minimal hyphae with no signs of invasiveness. The developed bioinspired nanoparticles serve as prospective bioactive nanocarrier candidates for pulmonary administration of VRZ in the management of IPA.

Aspergillus fumigatus cytochrome c impacts conidial survival during sterilizing immunity.

IMPORTANCE: Aspergillus fumigatus can cause a life-threatening infection known as invasive pulmonary aspergillosis (IPA), which is marked by fungus-attributable mortality rates of 20%-30%. Individuals at risk for IPA harbor genetic mutations or incur pharmacologic defects that impair myeloid cell numbers and/or function, exemplified by bone marrow transplant recipients, patients that receive corticosteroid therapy, or patients with chronic granulomatous disease (CGD). However, treatments for Aspergillus infections remain limited, and resistance to the few existing drug classes is emerging. Recently, the World Health Organization classified A. fumigatus as a critical priority fungal pathogen. Our cell death research identifies an important aspect of fungal biology that impacts susceptibility to leukocyte killing. Furthering our understanding of mechanisms that mediate the outcome of fungal-leukocyte interactions will increase our understanding of both the underlying fungal biology governing cell death and innate immune evasion strategies utilized during mammalian infection pathogenesis. Consequently, our studies are a critical step toward leveraging these mechanisms for novel therapeutic advances.

A Pathology-based Case Series of Influenza- and COVID-19-associated Pulmonary Aspergillosis: The Proof Is in the Tissue.

Rationale: Invasive pulmonary aspergillosis has emerged as a frequent coinfection in severe coronavirus disease (COVID-19), similarly to influenza, yet the clinical invasiveness is more debated. Objectives: We investigated the invasive nature of pulmonary aspergillosis in histology specimens of influenza and COVID-19 ICU fatalities in a tertiary care center. Methods: In this monocentric, descriptive, retrospective case series, we included adult ICU patients with PCR-proven influenza/COVID-19 respiratory failure who underwent postmortem examination and/or tracheobronchial biopsy during ICU admission from September 2009 until June 2021. Diagnosis of probable/proven viral-associated pulmonary aspergillosis (VAPA) was made based on the Intensive Care Medicine influenza-associated pulmonary aspergillosis and the European Confederation of Medical Mycology (ECMM) and the International Society for Human and Animal Mycology (ISHAM) COVID-19-associated pulmonary aspergillosis consensus criteria. All respiratory tissues were independently reviewed by two experienced pathologists. Measurements and Main Results: In the 44 patients of the autopsy-verified cohort, 6 proven influenza-associated and 6 proven COVID-19-associated pulmonary aspergillosis diagnoses were identified. Fungal disease was identified as a missed diagnosis upon autopsy in 8% of proven cases (n = 1/12), yet it was most frequently found as confirmation of a probable antemortem diagnosis (n = 11/21, 52%) despite receiving antifungal treatment. Bronchoalveolar lavage galactomannan testing showed the highest sensitivity for VAPA diagnosis. Among both viral entities, an impeded fungal growth was the predominant histologic pattern of pulmonary aspergillosis. Fungal tracheobronchitis was histologically indistinguishable in influenza (n = 3) and COVID-19 (n = 3) cases, yet macroscopically more extensive at bronchoscopy in influenza setting. Conclusions: A proven invasive pulmonary aspergillosis diagnosis was found regularly and with a similar histological pattern in influenza and in COVID-19 ICU case fatalities. Our findings highlight an important need for VAPA awareness, with an emphasis on mycological bronchoscopic work-up.

Serum amyloid P component is an essential element of resistance against Aspergillus fumigatus.

Serum amyloid P component (SAP, also known as Pentraxin 2; APCS gene) is a component of the humoral arm of innate immunity involved in resistance to bacterial infection and regulation of tissue remodeling. Here we investigate the role of SAP in antifungal resistance. Apcs-/- mice show enhanced susceptibility to A. fumigatus infection. Murine and human SAP bound conidia, activate the complement cascade and enhance phagocytosis by neutrophils. Apcs-/- mice are defective in vivo in terms of recruitment of neutrophils and phagocytosis in the lungs. Opsonic activity of SAP is dependent on the classical pathway of complement activation. In immunosuppressed mice, SAP administration protects hosts against A. fumigatus infection and death. In the context of a study of hematopoietic stem-cell transplantation, genetic variation in the human APCS gene is associated with susceptibility to invasive pulmonary aspergillosis. Thus, SAP is a fluid phase pattern recognition molecule essential for resistance against A. fumigatus.

Proven COVID-19-associated pulmonary aspergillosis in patients with severe respiratory failure.

BACKGROUND: An increasing number of reports have described the COVID-19-associated pulmonary aspergillosis (CAPA) as being a further contributing factor to mortality. Based on a recent consensus statement supported by international medical mycology societies, it has been proposed to define CAPA as possible, probable, or proven on the basis of sample validity and thus diagnostic certainty. Considering current challenges associated with proven diagnoses, there is pressing need to study the epidemiology of proven CAPA. METHODS: We report the incidence of histologically diagnosed CAPA in a series of 45 consecutive COVID-19 laboratory-confirmed autopsies, performed at Padova University Hospital during the first and second wave of the pandemic. Clinical data, laboratory data and radiological features were also collected for each case. RESULTS: Proven CAPA was detected in 9 (20%) cases, mainly in the second wave of the pandemic (7/17 vs. 2/28 of the first wave). The population of CAPA patients consisted of seven males and two females, with a median age of 74 years. Seven patients were admitted to the intensive care unit. All patients had at least two comorbidities, and concomitant lung diseases were detected in three cases. CONCLUSION: We found a high frequency of proven CAPA among patients with severe COVID-19 thus confirming at least in part the alarming epidemiological data of this important complication recently reported as probable CAPA.

Invasive pulmonary aspergillosis associated with viral pneumonitis.

The occurrence of invasive pulmonary aspergillosis (IPA) in critically ill patients with viral pneumonitis has increasingly been reported in recent years. Influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA) are the two most common forms of this fungal infection. These diseases cause high mortality in patients, most of whom were previously immunocompetent. The pathogenesis of IAPA and CAPA is still not fully understood, but involves viral, fungal and host factors. In this article, we discuss several aspects regarding IAPA and CAPA, including their possible pathogenesis, the use of immunotherapy, and future challenges.

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.

Invasive pulmonary aspergillosis with brain dissemination in an immunocompetent host

Invasive aspergillosis is an uncommon infection, which is mainly seen among immunocompromised patients. In recent years, cases of aspergillosis involving immunocompetent hosts are increasingly being reported. Herein, we report the case of a 27-year-old man with fever, productive cough, shortness of breath, and left hemiparesis. He had suffered trauma to his head 25 days prior. Imaging of the chest showed bilateral cavitary lesions in the lungs, and neuroimaging revealed a space-occupying lesion in the right frontoparietal cerebrum. He was suspected of having an abscess or metastasis. He died on day 3 of hospitalization, and an autopsy was performed. The autopsy revealed the cause of death to be invasive pulmonary aspergillosis, with brain dissemination. Invasive aspergillosis is uncommon in apparently immunocompetent individuals, and we discuss the autopsy findings in detail.

In vitro study on aspects of molecular mechanisms underlying invasive aspergillosis caused by gliotoxin and fumagillin, alone and in combination.

Gliotoxin (GT) and fumagillin (FUM) are mycotoxins most abundantly produced by Aspergillus fumigatus during the early stages of infection to cause invasive aspergillosis (IA). Therefore, we hypothesized that GT and FUM could be the possible source of virulence factors, which we put to test adopting in vitro monoculture and the novel integrated multiple organ co-culture (IdMOC) of A549 and L132 cell. We found that (i) GT is more cytotoxic to lung epithelial cells than FUM, and (ii) GT and FUM act synergistically to inflict pathology to the lung epithelial cell. Reactive oxygen species (ROS) is the master regulator of the cytotoxicity of GT, FUM and GT + FUM. ROS may be produced as a sequel to mitochondrial damage and, thus, mitochondria are both the source of ROS and the target to ROS. GT-, FUM- and GT + FUM-induced DNA damage is mediated either by ROS-dependent mechanism or directly by the fungal toxins. In addition, GT, FUM and GT + FUM may induce protein accumulation. Further, it is speculated that GT and FUM inflict epithelial damage by neutrophil-mediated inflammation. With respect to multiple organ cytotoxicity, GT was found to be cytotoxic at IC50 concentration in the following order: renal epithelial cells < type II epithelial cells < hepatocytes < normal lung epithelial cells. Taken together, GT and FUM alone and in combination contribute to exacerbate the damage of lung epithelial cells and, thus, are involved in the progression of IA.

Confirmed Invasive Pulmonary Aspergillosis and COVID-19: the value of postmortem findings to support antemortem management.

We present postmortem evidence of invasive pulmonary aspergillosis (IPA) in a patient with severe COVID-19. Autopsies of COVID-19 confirmed cases were performed. The patient died despite antimicrobials, mechanical ventilation, and vasopressor support. Histopathology and peripheral blood galactomannan antigen testing confirmed IPA. Aspergillus penicillioides infection was confirmed by nucleotide sequencing and BLAST analysis. Further reports are needed to assess the occurrence and frequency of IPA in SARS-CoV-2 infections, and how they interact clinically.

CT of invasive pulmonary aspergillosis (IPA) in cases with hematologic malignancy: Comparison of CT features in the group classified by the severity of neutropenia and underlying disease.

PURPOSE: To identify differences in the radiological findings of invasive pulmonary aspergillosis (IPA) among patients classified by severity of neutropenia, and differences in underlying disease. MATERIALS AND METHODS: We retrospectively reviewed computed tomography (CT) scans from the time of the diagnosis of IPA in 83 hematological malignancy patients with probable or proven IPA according to the EORTC-MSG criteria. We evaluated CT findings (radiological pattern, number of lesions, distribution, and presence of low attenuation area [LAA]), and compared the radiological findings of patients classified by degree of neutropenia with two different indicators (neutrophil count at the onset, and c-d-index) and underlying disease. RESULTS: Neutropenia at the onset of IPA was associated with an increased frequency of LAA (p < 0.05), especially in FN (p < 0.01). Cases with a c-d-index of ≧5500 showed an increased incidence of the angio-invasive pattern. In contrast, cases with a c-d-index of 0 showed an increased incidence of the airway-invasive pattern (p < 0.05). The airway-invasive pattern was more frequent in cases with MM, while the angio-invasive pattern was more frequent in cases with AML (p < 0.01). Lower-predominant distribution was more frequent and random distribution was less frequent in cases with AML, random distribution was more frequent and lower-predominant distribution was less frequent in cases with ALL, and upper-predominant distribution was more frequent in cases with MDS (p < 0.05). CONCLUSIONS: CT features of IPA vary according to the degree of neutropenia and underlying disease.

Three-Dimensional Light Sheet Fluorescence Microscopy of Lungs To Dissect Local Host Immune-Aspergillus fumigatus Interactions.

Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment, and progression of A. fumigatus infections are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are of paramount importance to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here, we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia, or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction, and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in complete intra-alveolar deposition, as about 80% of fungal growth occurred outside the alveolar space. Hence, characterization by LSFM is more rigorous than by previously used methods employing murine models of invasive pulmonary aspergillosis and pinpoints their strengths and limitations.IMPORTANCE The use of animal models of infection is essential to advance our understanding of the complex host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in the case of humans, mice need to suffer an immune imbalance in order to become susceptible to invasive pulmonary aspergillosis (IPA), the most serious infection caused by A. fumigatus There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis. However, the precise consequences of the use of each immunosuppressive model for the local immune populations and for fungal growth are not completely understood. Here, to pin down the scenarios involving commonly used IPA models, we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution. Our results will be valuable to optimize and refine animal models to maximize their use in future research.

Confirmed Invasive Pulmonary Aspergillosis and COVID-19: the value of postmortem findings to support antemortem management

Abstract We present postmortem evidence of invasive pulmonary aspergillosis (IPA) in a patient with severe COVID-19. Autopsies of COVID-19 confirmed cases were performed. The patient died despite antimicrobials, mechanical ventilation, and vasopressor support. Histopathology and peripheral blood galactomannan antigen testing confirmed IPA. Aspergillus penicillioides infection was confirmed by nucleotide sequencing and BLAST analysis. Further reports are needed to assess the occurrence and frequency of IPA in SARS-CoV-2 infections, and how they interact clinically.

The grim reaper evading modern medicine: aspergillosis, adenovirus, and Hodgkin lymphoma

Illustrative cases of diseases that are difficult to suspect and diagnose can serve as useful reminders. Invasive pulmonary aspergillosis and adenovirus hepatitis are two such diseases, both revealed by autopsy in this case of Hodgkin lymphoma refractory to chemotherapy treated with allogeneic hematopoietic stem cell transplantation complicated by these two fatal infections. This patient was cured of Hodgkin lymphoma, Clostridioides difficile colitis and thrombotic thrombocytopenic purpura using the marvels of modern medicine. This case illustrates many features of aspergillosis and adenovirus hepatitis, shows the value of autopsy in revealing diagnoses, and illustrates the limits of modern medicine, which should serve as a mental spur in our efforts to advance medical science, to try to defeat the numerous demons of disease, who seem to keep outwitting us.

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.

Two KTR Mannosyltransferases Are Responsible for the Biosynthesis of Cell Wall Mannans and Control Polarized Growth in Aspergillus fumigatus.

Fungal cell wall mannans are complex carbohydrate polysaccharides with different structures in yeasts and molds. In contrast to yeasts, their biosynthetic pathway has been poorly investigated in filamentous fungi. In Aspergillus fumigatus, the major mannan structure is a galactomannan that is cross-linked to the ß-1,3-glucan-chitin cell wall core. This polymer is composed of a linear mannan with a repeating unit composed of four α1,6-linked and α1,2-linked mannoses with side chains of galactofuran. Despite its use as a biomarker to diagnose invasive aspergillosis, its biosynthesis and biological function were unknown. Here, we have investigated the function of three members of the Ktr (also named Kre2/Mnt1) family (Ktr1, Ktr4, and Ktr7) in A. fumigatus and show that two of them are required for the biosynthesis of galactomannan. In particular, we describe a newly discovered form of α-1,2-mannosyltransferase activity encoded by the KTR4 gene. Biochemical analyses showed that deletion of the KTR4 gene or the KTR7 gene leads to the absence of cell wall galactomannan. In comparison to parental strains, the Δktr4 and Δktr7 mutants showed a severe growth phenotype with defects in polarized growth and in conidiation, marked alteration of the conidial viability, and reduced virulence in a mouse model of invasive aspergillosis. In yeast, the KTR proteins are involved in protein 0- and N-glycosylation. This study provided another confirmation that orthologous genes can code for proteins that have very different biological functions in yeasts and filamentous fungi. Moreover, in A. fumigatus, cell wall mannans are as important structurally as ß-glucans and chitin.IMPORTANCE The fungal cell wall is a complex and dynamic entity essential for the development of fungi. It allows fungal pathogens to survive environmental challenge posed by nutrient stress and host defenses, and it also is central to polarized growth. The cell wall is mainly composed of polysaccharides organized in a three-dimensional network. Aspergillus fumigatus produces a cell wall galactomannan whose biosynthetic pathway and biological functions remain poorly defined. Here, we described two new mannosyltransferases essential to the synthesis of the cell wall galactomannan. Their absence leads to a growth defect with misregulation of polarization and altered conidiation, with conidia which are bigger and more permeable than the conidia of the parental strain. This study showed that in spite of its low concentration in the cell wall, this polysaccharide is absolutely required for cell wall stability, for apical growth, and for the full virulence of A. fumigatus.

Deep sequencing profiles MicroRNAs related to Aspergillus fumigatus infection in lung tissues of mice.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is a severe opportunistic infection with high mortality in patients with compromised immunity. The full repertoire of microRNAs (miRNAs) involved in the regulation of IPA infection remains to be established. METHODS: We established a mouse IPA model and analyzed small RNA transcriptomes in lung tissues of immunodeficient IPA mice (IPA group) and matched immunodeficient control mice (control group) through next-generation sequencing. RESULTS: A total of 3759 known miRNAs were detected, in which 23 miRNAs were identified to be related to IPA. IPA-associated miRNAs include upregulated mmu-let-7b-3p, mmu-miR124-3p, mmu-miR21a-3p, mmu-miR29c-5p, mmu-miR3473b and mmu-miR3473e, and downregulated mmu-miR-150-3p and mmu-miR-503-5p. The expression levels of eight identified miRNAs were quantified in a validation cohort (n = 40) by qRT-PCR, and results revealed the same change patterns. MiRNA target prediction revealed that all IPA-related miRNAs possibly engage a cooperative regulation of key elements in the NF-kappa B signaling pathway. CONCLUSION: We conclude that deep-sequencing small RNAs can uncover miRNA pool-regulating IPA. Our results may lead to further understanding IPA pathogenesis and gain insight into the complexity and diversity of small RNA molecules that regulate immunodeficient IPA.

Aspergillus terreus Causing Probable Invasive Aspergillosis in a Patient with Cystic Fibrosis.

Aspergillus terreus may colonize the airways of patients with cystic fibrosis (CF). Whether this merits antifungal treatment is still unclear due to heterogeneous reports regarding its pathogenic potential. Although allergic manifestations are documented, invasive aspergillosis (IA) caused by A. terreus has not been described. We present here one case of probable IA caused by A. terreus, highlighting its role as an emerging agent of IA in CF patients. Voriconazole was effective, and the patient was healthy after 12 weeks of treatment. Serial determination of serum galactomannan level and periodic cultures from respiratory specimens may help in monitoring CF patients by identifying such pathogens. Further studies on the fungal species colonizing the CF airways are warranted, especially in India. This case report suggests A. terreus as a potential agent of IA which should not be ignored, particularly in this patient group.

Invasive Pulmonary Aspergillosis Complicated by Carbapenem-Resistant Pseudomonas aeruginosa Infection During Pembrolizumab Immunotherapy for Metastatic Lung Adenocarcinoma: Case Report and Review of the Literature.

The widespread use of T lymphocyte-associated antigen-4 (CTLA-4) and programmed death (PD)-1 and PD ligand-1 (PDL1)-targeted agents in cancer patients as immunotherapy has raised some issues on their safety profile. Regarding infectious complications, it has emerged that these compounds do not intrinsically increase susceptibility to opportunistic infections, which mainly correlate with the co-administration of systemic immunosuppressive therapy (high-dose corticosteroids and anti-tumor necrosis factors inhibitors) to cure immune-related adverse events (colitis, hepatitis, pneumonitis and pancreatitis), well-known complications of these targeted drugs. These observations lead experts' opinion to suggest primary anti-Pneumocystis prophylaxis in patients undergoing CTLA-4 and PD-1/PDL1 agents who will receive prednisone 20 mg daily for ≥ 4 weeks. Few data on invasive fungal infections in this context are available. We report here a case of probable invasive pulmonary aspergillosis (p-IPA) complicating first-line immunotherapy with pembrolizumab for metastatic lung cancer that was further aggravated by multidrug-resistant Pseudomonas aeruginosa superinfection of fungal cavities; the patient received concurrent systemic corticosteroid therapy as anti-edema treatment for cerebral metastases. Reviewing literature about Aspergillus diseases in subjects receiving CTLA-4 and PD-1 and PDL1-targeted agents, we found three cases of invasive aspergillosis and one case of exacerbation of chronic progressive pulmonary aspergillosis after nivolumab treatment; to the best of our knowledge, this is the first report of p-IPA complicating pembrolizumab immunotherapy. Briefly, in this new setting of biological/targeted drugs, waiting for growing clinical experience, we recommend a high level of alertness in diagnosing any infectious complications.