Immunopathogenesis of Aspergillosis

Aspergillus species are ubiquitous saprophytes and opportunistic pathogens causing wide spectrum of diseases in humans depending on the host immune status. Following pathogen entry, various soluble bronchopulmonary factors enhance conidial clearance. However, due to virulence factors and poor host immune response Aspergillus conidia bind and damage the airway epithelium. The host immune cells like neutrophils and macrophages recognise Aspergillus spp. through various pathogen recognition receptors and form reactive oxygen species which mediate conidial killing. Neutrophils also attack extracellular hyphae by oxidative attack, non-oxidative granule proteins and neutrophil extracellular traps. In case of adaptive immunity, Th1 cells are crucial sources of IFN-γ mediated protective immunity. The Th17 also display a highly pro-inflammatory which is counterbalanced by a Treg cell. B cells and antibodies also enhance fungal clearance although excessive IgE production may result in atopy. The immune responses are influenced by changes in production of short-chain fatty acids by the gut microbiome which primes cells toward Th2 responses, and this is synchronized by the Innate lymphoid cells. This review provides comprehensive knowledge of various virulence factors of Aspergillus, antifungal host defences including innate and humoral immune response and regulation of host immunity by microbiome

Aerosolizable Lipid-Nanovesicles Encapsulating Voriconazole Effectively Permeate Pulmonary Barriers and Target Lung Cells

The entire world has recently been witnessing an unprecedented upsurge in microbial lung infections. The major challenge encountered in treating the same is to ensure the optimum drug availability at the infected site. Aerosolization of antimicrobials, in this regard, has shown immense potential owing to their localized and targeted effect. Efforts, therefore, have been undertaken to systematically develop lung-phosphatidylcholine-based lipid nanovesicles of voriconazole for potential management of the superinfections like aspergillosis. LNVs, prepared by thin-film hydration method, exhibited a globule size of 145.4 ± 19.5 nm, polydispersity index of 0.154 ± 0.104 and entrapment efficiency of 71.4 ± 2.2% with improved in vitro antifungal activity. Aerodynamic studies revealed a microdroplet size of ≤5 μm, thereby unraveling its promise to target the physical barrier of lungs effectively. The surface-active potential of LNVs, demonstrated through Langmuir-Blodgett troughs, indicated their ability to overcome the biochemical pulmonary surfactant monolayer barrier, while the safety and uptake studies on airway-epithelial cells signified their immense potential to permeate the cellular barrier of lungs. The pharmacokinetic studies showed marked improvement in the retention profile of voriconazole in lungs following LNVs nebulization compared to pristine voriconazole. Overall, LNVs proved to be safe and effective delivery systems, delineating their distinct potential to efficiently target the respiratory fungal infections

Nebulised surface-active hybrid nanoparticles of voriconazole for pulmonary Aspergillosis demonstrate clathrin-mediated cellular uptake, improved antifungal efficacy and lung retention

Abstract: Background: Incidence of pulmonary aspergillosis is rising worldwide, owing to an increased population of immunocompromised patients. Notable potential of the pulmonary route has been witnessed in antifungal delivery due to distinct advantages of direct lung targeting and first-pass evasion. The current research reports biomimetic surface-active lipid-polymer hybrid (LPH) nanoparticles (NPs) of voriconazole, employing lung-specific lipid, i.e., dipalmitoylphosphatidylcholine and natural biodegradable polymer, i.e., chitosan, to augment its pulmonary deposition and retention, following nebulization. Results: The developed nanosystem exhibited a particle size in the range of 228–255 nm and drug entrapment of 45–54.8%. Nebulized microdroplet characterization of NPs dispersion revealed a mean diameter of ≤ 5 μm, corroborating its deep lung deposition potential as determined by next-generation impactor studies. Biophysical interaction of LPH NPs with lipid-monolayers indicated their surface-active potential and ease of intercalation into the pulmonary surfactant membrane at the air-lung interface. Cellular viability and uptake studies demonstrated their cytocompatibility and time-and concentration-dependent uptake in lung-epithelial A549 and Calu-3 cells with clathrin-mediated internalization. Transepithelial electrical resistance experiments established their ability to penetrate tight airway Calu-3 monolayers. Antifungal studies on laboratory strains and clinical isolates depicted their superior efficacy against Aspergillus species. Pharmacokinetic studies revealed nearly 5-, 4- and threefolds enhancement in lung AUC, Tmax, and MRT values, construing significant drug access and retention in lungs. Conclusions: Nebulized LPH NPs were observed as a promising solution to provide effective and safe therapy for the management of pulmonary aspergillosis infection with improved patient compliance and avoidance of systemic side-effects

ABC Transporter Genes Show Upregulated Expression in Drug Resistant Clinical Isolates of Candida auris : a Genome-Wide Characterization of ATP-Binding Cassette (ABC) Transporter Genes

Funding This work was supported by the ICMR (AMR/149/2018-ECD-II) and DBT (BT/PR14117/BRB/10/1420/2015) to RP. AKM appreciates the support by research grant EMR/2016/001927 and DST PURSE II from the Department of Science and Technology (IN). MW was grateful for a Senior Research Fellowship from the University Grant Commission. NG acknowledges the Wellcome Trust support of a Senior Investigator (101873/Z/13/Z), Collaborative (200208/A/15/Z), and Strategic Awards (097377/Z11/Z), and the MRC Centre for Medical Mycology (MR/N006364/1). AJM was supported by the University of Aberdeen studentship. Work in AL’s laboratory was supported by the Wellcome Trust (212524/Z/18/Z) and the Medical Research Council (MRC) Centre for Medical Mycology at the University of Aberdeen (MR/P501955/1 and MR/N006364/1). Acknowledgments We thank the Centre for Genome Enabled Biology and Medicine at the University of Aberdeen (E. Collie-Duguid and S. Shaw) for sequencing and support with genome analysis.Peer reviewedPublisher PD

High Resolution Genotyping of Clinical Aspergillus flavus Isolates from India Using Microsatellites

Contains fulltext : 124312.pdf (publisher's version ) (Open Access)BACKGROUND: Worldwide, Aspergillus flavus is the second leading cause of allergic, invasive and colonizing fungal diseases in humans. However, it is the most common species causing fungal rhinosinusitis and eye infections in tropical countries. Despite the growing challenges due to A. flavus, the molecular epidemiology of this fungus has not been well studied. We evaluated the use of microsatellites for high resolution genotyping of A. flavus from India and a possible connection between clinical presentation and genotype of the involved isolate. METHODOLOGY/PRINCIPAL FINDINGS: A panel of nine microsatellite markers were selected from the genome of A. flavus NRRL 3357. These markers were used to type 162 clinical isolates of A. flavus. All nine markers proved to be polymorphic displaying up to 33 alleles per marker. Thirteen isolates proved to be a mixture of different genotypes. Among the 149 pure isolates, 124 different genotypes could be recognized. The discriminatory power (D) for the individual markers ranged from 0.657 to 0.954. The D value of the panel of nine markers combined was 0.997. The multiplex multicolor approach was instrumental in rapid typing of a large number of isolates. There was no correlation between genotype and the clinical presentation of the infection. CONCLUSIONS/SIGNIFICANCE: There is a large genotypic diversity in clinical A. flavus isolates from India. The presence of more than one genotype in clinical samples illustrates the possibility that persons may be colonized by multiple genotypes and that any isolate from a clinical specimen is not necessarily the one actually causing infection. Microsatellites are excellent typing targets for discriminating between A. flavus isolates from various origins

Nucleic-Acid-Based Molecular Fungal Diagnostics: A Way to a Better Future

The world has seen a tremendous increase in the number of fungal infections during the past two decades. Recently, the World Health Organisation released the pathogen priority list for fungal infections, signifying the importance of these infections in the fields of research and public health. Microbiology laboratories demand an upgrade in the diagnostic system to keep up with the increased burden of these infections. Diagnosis of fungal infections using conventional techniques has always faced limitations in terms of specificity, sensitivity, and turnaround time. Although these methods are the core pillars of the diagnosis, there is an increased need for molecular approaches. Molecular techniques have revolutionised the field of fungal diagnostics. The diverse array of molecular techniques, including techniques like Polymerase Chain Reaction (PCR), have emerged as a cornerstone in fungal diagnostics. Molecular techniques have transformed fungal diagnostics, providing powerful tools for the rapid and accurate identification of pathogens. As these technologies continue to evolve, their integration into routine clinical practice holds the promise of improving patient outcomes through timely and targeted antifungal interventions. This review will cover the molecular approaches involved in fungal diagnostics, moving from the basic techniques to the advanced-level nucleic-acid-based molecular approaches providing a high throughput and decreased turnaround time for the diagnosis of serious fungal infections

The Comparative Evaluation of the Fujifilm Wako β-Glucan Assay and Fungitell Assay for Diagnosing Invasive Fungal Disease

Serum 1,3-β-d-glucan(BDG) is a broad fungal biomarker for invasive fungal disease (IFD). More data is still required to support the Fujifilm Wako assay as a valuable alternative to the widely used Fungitell assay. We included archived serum samples from 157 individuals (97 cases; 33-IA, 64-IC, and 60 controls) for the comparative performance evaluation of the Fungitell assay and the Fujifilm Wako assay for IFD diagnosis. The BDG value was significantly higher in patients with IFD vs. controls (70.79 pg/mL vs. 3.03 pg/mL, p: 0.0002). An area under the curve (AUC) for the IFD, IC, and IA diagnosis was 0.895, 0.910, and 0.866, respectively, for the Fujifilm Wako assay. Based on the highest Youden’s index (0.667), a cutoff of 5 pg/mL was selected as the optimum for the Fujifilm Wako assay with good sensitivity (79.4%), specificity (88.3%) and agreement (84.7%, Cohen’s k:0.691) with the Fungitell assay. The mean run-time of the Fujifilm Wako assay was 70.12 min, and real-time observation allowed earlier time to result in Fujifilm Wako vs. Fungitell assay (37 vs. 120 min, p: < 0.0001). Thus, our findings support the diagnostic value of the Fujifilm Wako assay for the diagnosis of IFD. However, there is still a need to validate diagnostic protocols to optimize their use in multi-centre studies with different patient groups

Expression and Purification along with Evaluation of Serological Response and Diagnostic Potential of Recombinant Sap2 Protein from <i>C. parapsilosis</i> for Use in Systemic Candidiasis

Systemic candidiasis is the fourth most common bloodstream infection in ICU patients worldwide. Although C. albicans is a predominant species causing systemic candidiasis, infections caused by non-albicans Candida (NAC) species are increasingly becoming more prevalent globally along with the emergence of drug resistance. The diagnosis of systemic candidiasis is difficult due to the absence of significant clinical symptoms in patients. We investigated the diagnostic potential of recombinant secreted aspartyl proteinase 2 (rSap2) from C. parapsilosis for the detection of Candida infection. The rSap2 protein was successfully cloned, expressed and purified using Ni-NTA chromatography under denaturing conditions using an E. coli-based prokaryotic expression system, and refolded using a multi-step dialysis procedure. Structural analysis by CD and FTIR spectroscopy revealed the refolded protein to be in its near native conformation. Immunogenicity analysis demonstrated the rSap2 protein to be highly immunogenic as evident from significantly high titers of Sap2-specific antibodies in antigen immunized Balb/c mice, compared to sham-immunized controls. The diagnostic potential of rSap2 protein was evaluated using immunoblotting and ELISA assays using proven candidiasis patient serum and controls. Immunoblotting results indicate that reactivity to rSap2 was specific to candidiasis patient sera with no cross reactivity observed in healthy controls. Increased levels of anti-Sap2-specific Ig, IgG and IgM antibodies were observed in candidiasis patients compared to controls and was similar in sensitivity obtained when whole Candida was used as coating antigen. In summary, the rSap2 protein from C. parapsilosis has the potential to be used in the diagnosis of systemic candidiasis, providing a rapid, convenient, accurate and cost-effective strategy