Treatment Of Cryptococcosis Research Articles

Discovery of New Tricyclic Oxime Sampangine Derivatives as Potent Antifungal Agents for the Treatment of Cryptococcosis and Candidiasis.

Cryptococcus neoformans (C. neoformans) and Candida albicans (C. albicans) are classified as the critical priority groups among the pathogenic fungi, highlighting the urgent need for developing more effective antifungal therapies. On the basis of antifungal natural product sampangine, herein, a series of tricyclic oxime and oxime ether derivatives were designed. Among them, compound WZ-2 showed excellent inhibitory activity against C. neoformans (MIC80 = 0.016 μg/mL) and synergized with fluconazole to treat resistant C. albicans (FICI = 0.078). Interestingly, compound WZ-2 effectively inhibited virulence factors (e.g., capsule, biofilm, and yeast-to-hypha morphological transition), suggesting the potential to overcome drug resistance. In a mouse model of cryptococcal meningitis, compound WZ-2 (5 mg/kg) effectively reduced the brain C. neoformans H99 burden. Furthermore, compound WZ-2 alone and its combination with fluconazole also significantly reduced the kidney burden of the drug-resistant strain (0304103) and sensitive strain (SC5314) of C. albicans.

Novel combinatorial approach: Harnessing HIV protease inhibitors to enhance amphotericin B's antifungal efficacy in cryptococcosis.

Cryptococcosis is a fungal infection that is becoming increasingly prevalent worldwide, particularly among individuals with compromised immune systems, such as HIV patients. Amphotericin B (AmB) is the first-line treatment mainly combined with flucytosine. The scarcity and the prohibitive cost of this regimen urge the use of fluconazole as an alternative, leading to increased rates of treatment failure and relapses. Therefore, there is a critical need for efficient and cost-effective therapy to enhance the efficacy of AmB. In this study, we evaluated the efficacy of the HIV protease inhibitors (PIs) to synergize the activity of AmB in the treatment of cryptococcosis. Five PIs (ritonavir, atazanavir, saquinavir, lopinavir, and nelfinavir) were found to synergistically potentiate the killing activity of AmB against Cryptococcus strains with ƩFICI ranging between 0.09 and 0.5 against 20 clinical isolates. This synergistic activity was further confirmed in a time-kill assay, where different AmB/PIs combinations exhibited fungicidal activity within 24 hrs. Additionally, PIs in combination with AmB exhibited an extended post-antifungal effect on treated cryptococcal cells for approximately 10 hrs compared to 4 hours with AmB alone. This promising activity against cryptococcal cells did not exhibit increased cytotoxicity towards treated kidney cells, ruling out the risk of drug combination-induced nephrotoxicity. Finally, we evaluated the efficacy of AmB/PIs combinations in the Caenorhabditis elegans model of cryptococcosis, where these combinations significantly reduced the fungal burden of the treated nematodes by approximately 2.44 Log10 CFU (92.4%) compared to the untreated worms and 1.40 Log10 ((39.4%) compared to AmB alone. The cost-effectiveness and accessibility of PIs in resource-limited geographical areas compared to other antifungal agents, such as flucytosine, make them an appealing choice for combination therapy.

Aneuploidy underlies brefeldin A-induced antifungal drug resistance in Cryptococcus neoformans.

Cryptococcus neoformans is at the top of the list of "most wanted" human pathogens. Only three classes of antifungal drugs are available for the treatment of cryptococcosis. Studies on antifungal resistance mechanisms are limited to the investigation of how a particular antifungal drug induces resistance to a particular drug, and the impact of stresses other than antifungals on the development of antifungal resistance and even cross-resistance is largely unexplored. The endoplasmic reticulum (ER) is a ubiquitous subcellular organelle of eukaryotic cells. Brefeldin A (BFA) is a widely used chemical inducer of ER stress. Here, we found that both weak and strong selection by BFA caused aneuploidy formation in C. neoformans, mainly disomy of chromosome 1, chromosome 3, and chromosome 7. Disomy of chromosome 1 conferred cross-resistance to two classes of antifungal drugs: fluconazole and 5-flucytosine, as well as hypersensitivity to amphotericin B. However, drug resistance was unstable, due to the intrinsic instability of aneuploidy. We found overexpression of AFR1 on Chr1 and GEA2 on Chr3 phenocopied BFA resistance conferred by chromosome disomy. Overexpression of AFR1 also caused resistance to fluconazole and hypersensitivity to amphotericin B. Furthermore, a strain with a deletion of AFR1 failed to form chromosome 1 disomy upon BFA treatment. Transcriptome analysis indicated that chromosome 1 disomy simultaneously upregulated AFR1, ERG11, and other efflux and ERG genes. Thus, we posit that BFA has the potential to drive the rapid development of drug resistance and even cross-resistance in C. neoformans, with genome plasticity as the accomplice.

Cryptococcosis in kidney transplant recipients: Current understanding and practices.

Cryptococcosis is the third most commonly occurring invasive fungal disease in solid organ transplant recipients (SOT). It is caused by encapsulated yeast, Cryptococcus species, predominantly Cryptococcus neoformans and Cryptococcus gattii. Though kidney transplant recipients are at the lowest risk of cryptococcosis when compared to other solid organ transplant recipients such as lung, liver or heart, still this opportunistic infection causes significant morbidity and mortality in this subset of patients. Mortality rates with cryptococcosis range from 10%-25%, while it can be as high as 50% in SOT recipients with central nervous system involvement. The main aim of diagnosis is to find out if there is any involvement of the central nervous system in disseminated disease or whether there is only localized pulmonary involvement as it has implications for both prognostication and treatment. Detection of cryptococcal antigen (CrAg) in cerebrospinal fluid or plasma is a highly recommended test as it is more sensitive and specific than India ink and fungal cultures. The CrAg lateral flow assay is the single point of care test that can rapidly detect cryptococcal polysaccharide capsule. Treatment of cryptococcosis is challenging in kidney transplant recipients. Apart from the reduction or optimization of immunosuppression, lipid formulations of amphotericin B are preferred as induction antifungal agents. Consolidation and maintenance are done with fluconazole; carefully monitoring its interactions with calcineurin inhibitors. This review further discusses in depth the evolving developments in the epidemiology, pathogenesis, diagnostic assays, and management approach of cryptococcosis in kidney transplant recipients.

Novel antimicrobial peptide DvAMP serves as a promising antifungal agent against Cryptococcus neoformans

Cryptococcus neoformans is an important opportunistic human fungal pathogen that causes cryptococcosis in immunocompromised patients. However, the number of drugs for the treatment of cryptococcosis is restricted, and the development of novel antifungal drugs and innovative strategies for the treatment of cryptococcosis is urgently needed. In this study, we validated that DvAMP is a novel antimicrobial peptide with antimicrobial activity and that it was obtained by pre-screening from the UniProt database of more than three million unknown functional sequences based on the quantitative structure–activity relationships (QSARs) protocol (http://www.chemoinfolab.com/antifungal). The peptide exhibited satisfactory biosafety and physicochemical properties, and relatively rapid fungicidal activity against C. neoformans. Meanwhile, DvAMP was able to inhibit the static biofilm of C. neoformans and cause a reduction in the thickness of the capsule. In addition, DvAMP exerts antifungal effects through membrane-mediated mechanisms (membrane permeability and depolarization) and mitochondrial dysfunction, involving a hybrid multi-hit mechanism. Furthermore, by using the C. neoformans-Galleria mellonella infection model, we demonstrated that DvAMP has significant therapeutic effects in vivo and that it significantly reduces the mortality and fungal burden of infected larvae. These results suggest that DvAMP may be a potential antifungal drug candidate for the treatment of cryptococcosis.

Antifungal Peptide SP1 Damages Polysaccharide Capsule of Cryptococcus neoformans and Enhances Phagocytosis of Macrophages.

Cryptococcus neoformans is a fungal pathogen which causes nearly half a million deaths worldwide each year. Under host-relevant conditions, it produces a characteristic polysaccharide capsule. The polysaccharide capsule is one of the main virulence factors of C. neoformans, which involves antiphagocytosis and immune responses of the host to cause a lack of an immune. Meanwhile, the polysaccharide capsule is a promising drug target because of the absence of analogs in the host. Here, we demonstrate that antifungal peptide SP1, which is derived from the N terminus of Saccharomyces cerevisiae GAPDH (glyceraldehyde-3-phosphate dehydrogenase), disrupts the polysaccharide capsule of C. neoformans H99. The mechanism is possibly due to the interaction of SP1 with glucuronoxylomannan (GXM). Disruption of the polysaccharide capsule enhances the adhesion and phagocytosis of C. neoformans H99 by macrophages and reduces the replication of C. neoformans H99 within macrophages. Additionally, SP1 exhibits antifungal activity against cryptococcal biofilms associated with the capsular polysaccharides. These findings suggest the potential of SP1 as a drug candidate for the treatment of cryptococcosis. IMPORTANCE C. neoformans is an opportunistic pathogen that causes invasive infections with a high mortality rate. Currently, the clinical drugs available for the treatment of cryptococcosis are limited to amphotericin B, azoles, and flucytosine. Amphotericin is nephrotoxic, and the widespread use of azoles and 5-flucytosine has led to a rapid development of drug resistance in C. neoformans. There is an urgent need to develop new and effective anticryptococcal drugs. Targeting virulence factors is a novel strategy for developing antifungal drugs. The antifungal peptide SP1 is capable of disrupting the polysaccharide capsule, which is a principal virulence factor of C. neoformans. Studying the mechanism by which SP1 damages the polysaccharide capsule and investigating the potential benefits of SP1 in removing C. neoformans from the host provides baseline data to develop a therapeutic strategy against refractory cryptococcal infections. This strategy would involve both inhibiting virulence factors and directly killing C. neoformans cells.

In vitro and in vivo synergistic effects of hydroxychloroquine and itraconazole on Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that can cause life-threatening invasive fungal infections. As its prevalence and drug resistance continue to rise, cryptococcosis requires new treatment options. Tapping into the potential antifungal effects of traditional drugs or combination therapy has become one of the options. This study was the first to examine the interaction of hydroxychloroquine (HCQ) and itraconazole (ITR) on Cryptococcus neoformans in vitro and in vivo. Our results showed that HCQ alone and in combination with ITR exhibited antifungal activity against C. neoformans planktonic cells. When HCQ was combined with ITR, the minimal inhibitory concentration (MIC) value of HCQ decreased to 32μg/mL, and the MIC value of ITR decreased from 0.25μg/mL to 0.06-0.25μg/mL. The time-killing curve showed that the combined application of HCQ and ITR significantly shortened the killing time, dynamically defining the antifungal activity. The minimum biofilm clearance concentration (MBEC) of HCQ was only 32μg/mL, which was significantly lower than the MIC of HCQ for planktonic cells. When combined with ITR, the MBEC of ITR decreased from 128μg/mL to 2-1μg/mL, and the MBEC of HCQ decreased from 32μg/mL to 4μg/mL, indicating a synergistic antifungal biofilm effect. In comparison to ITR alone, the combination of HCQ and ITR treatment increased the survival of C. neoformans-infected Galleria mellonella larvae and decreased the fungal burden of infected larvae. Mechanistic investigations revealed that HCQ might damage C. neoformans cell membranes, impact the structure of fungal cells, cause extracellular material leakage, and have a potent affinity for attaching to the C. neoformans genomic DNA. In conclusion, HCQ has potential clinical application in the treatment of cryptococcosis.

Factors Influencing the Nitrogen-Source Dependent Flucytosine Resistance in Cryptococcus Species.

Flucytosine (5-FC) is an antifungal agent commonly used for treatment of cryptococcosis and several other systemic mycoses. In fungi, cytosine permease and cytosine deaminase are known major players in flucytosine resistance by regulating uptake and deamination of 5-FC, respectively. Cryptococcus species have three paralogs each of cytosine permease (FCY2, FCY3, and FCY4) and cytosine deaminase (FCY1, FCY5 and FCY6). As in other fungi, we found FCY1 and FCY2 to be the primary cytosine deaminase and permease gene, respectively, in C. neoformans H99 (VNI), C. gattii R265 (VGIIa) and WM276 (VGI). However, when various amino acids were used as the sole nitrogen source, C. neoformans and C. gattii diverged in the function of FCY3 and FCY6. Though there was some lineage-dependent variability, the two genes functioned as the secondary permease and deaminase, respectively, only in C. gattii when the nitrogen source was arginine, asparagine, or proline. Additionally, the expression of FCY genes, excluding FCY1, was under nitrogen catabolic repression in the presence of NH4. Functional analysis of GAT1 and CIR1 gene deletion constructs demonstrated that these two genes regulate the expression of each permease and deaminase genes individually. Furthermore, the expression levels of FCY3 and FCY6 under different amino acids corroborated the 5-FC susceptibility in fcy2Δ or fcy1Δ background. Thus, the mechanism of 5-FC resistance in C. gattii under diverse nitrogen conditions is orchestrated by two transcription factors of GATA family, cytosine permease and deaminase genes. IMPORTANCE 5-FC is a commonly used antifungal drug for treatment of cryptococcosis caused by Cryptococcus neoformans and C. gattii species complexes. When various amino acids were used as the sole nitrogen source for growth, we found lineage dependent differences in 5-FC susceptibility. Deletion of the classical cytosine permease (FCY2) and deaminase (FCY1) genes caused increased 5-FC resistance in all tested nitrogen sources in C. neoformans but not in C. gattii. Furthermore, we demonstrate that the two GATA family transcription factor genes GAT1 and CIR1 are involved in the nitrogen-source dependent 5-FC resistance by regulating the expression of the paralogs of cytosine permease and deaminase genes. Our study not only identifies the new function of paralogs of the cytosine permease and deaminase and the role of their regulatory transcription factors but also denotes the differences in the mechanism of 5-FC resistance among the two etiologic agents of cryptococcosis under different nitrogen sources.

Off-label treatments as potential accelerators in the search for the ideal antifungal treatment of cryptococcosis.

Cryptococcosis is an opportunistic mycosis that mainly affects immunosuppressed patients. The treatment is a combination of three antifungal agents: amphotericin B, 5-flucytosine and fluconazole. However, these drugs have many disadvantages, such as high nephrotoxicity, marketing bans in some countries and fungal resistance. One of the solutions to find possible new drugs ispharmacological repositioning. This work presents repositioned drugs as an alternative for new antifungal therapies for cryptococcosis. All the studies here were performed in vitro or in animal models, except for sertraline, which reached phase III in humans. There is still no pharmacological repositioning approval for cryptococcosis in humans, though this review shows the potential of repurposing as a rapid approach to finding new agents to treat cryptococcosis.

RNAi machinery regulates nutrient metabolism and fluconazole resistance in the pathogenic fungus Cryptococcus deneoformans.

The RNAi machinery has been extensively studied in plant and animal cells for their crucial roles in the regulation of genome function. However, the potential roles of RNAi in controlling fungal growth and development have been poorly studied, especially in the basidiomycetous yeast Cryptococcus deneoformans. To characterize the biological functions of RNAi in the pathogenic fungus, a comparative analysis of mRNA profiles using high-throughput sequencing technology was performed for the wild type and the RNAi mutants of C. deneoformans. The results revealed a clear difference in the expression of genes associated with metabolic processes in the RNAi mutants. Besides, the growth under nutrient-limited conditions was significantly reduced in the ago2Δ mutant, suggesting the essential roles of Ago2 in nutrient metabolism. Further investigations revealed the differentially expressed transporters in the RNAi mutants, in which transporters involved in fluconazole efflux were significantly up-regulated. More importantly, on account of the upregulated transporters, RNAi mutant strains developed resistance to fluconazole. By disrupting AFR1 gene using the 'suicide' CRISPR-Cas9 system, we verified that the upregulated ABC transporter Afr1 in the RNAi mutants contributed to the fluconazole resistance. In summary, our data demonstrate that in C. deneoformans the RNAi pathway participates in nutrient metabolism and plays a role in the repression of fluconazole resistance, which provides a deep insight into RNAi mechanisms in Cryptococcus and brings great hints for the clinical treatment of cryptococcosis.

Pulmonary Cryptococcosis.

Pulmonary cryptococcosis describes an invasive lung mycosis caused by Cryptococcus neoformans or Cryptococcus gattii complex. It is often a high-consequence disease in both immunocompromised and immunocompetent populations, and may be misdiagnosed as pulmonary malignancy, leading to a delay in therapy. Epidemiology follows that of cryptococcal meningoencephalitis, with C. gattii infection more common in certain geographic regions. Diagnostic tools include histopathology, microscopy and culture, and the detection of cryptococcal polysaccharide antigen or Cryptococcus-derived nucleic acids. All patients with lung cryptococcosis should have a lumbar puncture and cerebral imaging to exclude central nervous system disease. Radiology is key, both as an adjunct to laboratory testing and as the initial means of detection in asymptomatic patients or those with non-specific symptoms. Pulmonary cryptococcomas (single or multiple) may also be associated with disseminated disease and/or cryptococcal meningitis, requiring prolonged treatment regimens. Optimal management for severe disease requires extended induction (amphotericin B and flucytosine) and consolidation therapy (fluconazole) with close clinical monitoring. Susceptibility testing is of value for epidemiology and in regions where relatively high minimum inhibitory concentrations to azoles (particularly fluconazole) have been noted. Novel diagnostic tools and therapeutic agents promise to improve the detection and treatment of cryptococcosis, particularly in low-income settings where the disease burden is high.

P379 Antifungal activity of α defensins 5 like peptide against Cryptococcus neoformans var. grubii and in silico designing of its peptidomimetics

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesCombinatorial and retroviral therapies have been found effective in the treatment of cryptococcosis, but safer and more potent antifungal therapeutics are the need of the hour. In this regard, the natural antifungal peptides (AFPs), the essential components of the innate immune system have drawn the attention of researchers and drug developers. AFPs are 12-50 amino-acids-long host defense peptides of cationic nature. Although AFPs have certain limitations like low stability, susceptibility to proteolysis, loss of activity in the presence of metal ions and salts, short half-lives, and peptide aggregation in oral formulation, but these can be addressed by designing and synthesizing the peptidomimetics.MethodologyThe whole blood of the rat was collected by cardiac puncture. The blood was mixed with 0.83% aqueous ammonium chloride. The neutrophils were separated from lysed WBCs by centrifugation. The purification of the peptide was carried out by SDS-PAGE, size exclusion, cation exchange chromatography, and HRLCMS. The MSA of the characterized peptide was performed using a T-coffee program. The in vitro antifungal activity of crude was tested against C. n. grubii (ATCC 6352) using flow cytometry. The structure of peptides was altered through ChemDraw by replacing the elements with their bioisosteres to form mimetics. The peptide and its mimetics were docked upon the target of C. n. grubii, i.e., O-acetyl transferase (modeled through Phyre2 web server) using PatchDock, and FireDock web servers.ResultsThe peptide isolated from rat PMNs had a molecular weight of 3 kDa. It has lysine and arginine which are the characteristic residues of an antifungal peptide. The eluted peptide has the highest (83%) similarity with α defensins 5 (accession no NP_001013071 XP_214 386). The mild antifungal activity of peptide was evident against C. n. grubii. The structure of O-acetyl transferase was modeled with 100% confidence and 92% query coverage (Table 1). The mimetic 1 (M1) showed the best interaction with O-acetyl transferase with −54.47 Kcal/mol binding energy and 4 hydrogen bonds (Table 2).ConclusionThe designed M1 of α defensins 5 like peptide could be a promising lead in antifungal drug development. As mimetic 1 was designed using an in-silico approach, its synthesis and wet lab validation are desirable.

P092 Otilonium bromide is a potent antifungal agent against fluconazole- and flucytosine-resistant Cryptococcus neoformans strains

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectives Cryptococcus neoformans is a worldwide threat causing global pulmonary and systemic infections in humans. However, only three drugs are available for the treatment of cryptococcosis: fluconazole, amphotericin B, and flucytosine. Drug repurposing, the process of using a drug for an indication different from the initial indication, is an emerging approach in the discovery of new antifungal drugs. We've set out to identify repurposable drugs for cryptococossis.MethodsWe screened the United States FDA-approved drugs for antifungal activity against C. albicans lab strain SC5314. We tested the antifungal profile against major human fungal pathogens including C. neoformans. In order to uncover the mechanism of action against C. neoformans, lab strain H99 was used to evolve drug resistant adaptors. Next-generation sequencing technology was used to investigate the genome change of drug resistant adaptors. SNP calling was also performed to identify possible mutations causing drug resistance.ResultsHere we found that otilonium bromide (OTB), which is extensively used to treat patients affected by the irritable bowel syndrome, had broad-spectrum antifungal activity. OTB was active against fluconaozle-resistant and flucotysine-resistant C. neoformans strains. Furthermore, we found resistance to OTB was mostly due to duplication of chromosome 6. Further work will be on the identification of the Candidate gene on chromosome 6 which is required OTB resistance.ConclusionThis study highlights the potential application of OTB as a new antifungal drug against C. neoformans strains susceptible or resistant to commonly used antifungal drugs.

S5.4d Agrochemicals potentiate multidrug resistance and alter virulence in Cryptococcus neoformans via hitch-hiking effect of aneuploidy

S5.4 Free oral paper session, September 22, 2022, 3:00 PM - 4:30 PMObjectives Cryptococcus neoformans is an environmental fungal pathogen causing fatal pulmonary and disseminated infections in humans, especially in AIDS patients. However, only three antifungal drugs are available for the treatment of cryptococcosis: fluconazole, amphotericin B, and flucytosine. C. neoformans is worldwide distributed in nature, but environmental factors influencing antifungal drug susceptibility are largely unknown.MethodsWe tested the antifungal effect of agrochemicals against C. neoformans lab strain H99. We obtained drug-resistant adaptors. We sequenced the adaptors and got a list of adaptors bearing unique aneuploidy. We asked if aneuploidy caused multidrug resistance to antifungal drugs and if aneuploidy altered virulence in vitro and in vivo. We also investigated the molecular mechanism of aneuploidy-mediated multidrug resistance.ResultsHere we found tebuconazole and benomyl, belonging to two distinct classes of agrochemicals, could inhibit the growth of H99. However, H99 could gain resistance to both drugs mainly via aneuploidy. Importantly, particular aneuploidy also caused multidrug resistance to fluconazole, amphotericin B, and flucytosine. Furthermore, aneuploidy also altered virulence in vitro and in vivo. We found copy number variation of genes on the aneuploid chromosome caused a proportional increase in transcript and protein levels.ConclusionTherefore, we posit agrochemicals could enhance multidrug resistance and alter the virulence of C. neoformans via the hitch-hiking effect of aneuploidy.

Phytochemical Analysis and Antifungal Potentiating Activity of Extracts from Loquat (Eriobotrya japonica) against Cryptococcus neoformans Clinical Isolates.

Eriobotrya japonica (loquat) has been used in African traditional medicine with numerous beneficial health effects. The extracts from loquat contain several bioactive compounds with a plethora of pharmacological properties. However, a scientific study on the activity against the aetiological agent of cryptococcosis has not yet been reported. Therefore, this study aimed to investigate the antifungal potential of various extracts from Eriobotrya japonica against clinical isolates of Cryptococcus neoformans. Quantitative and qualitative phytochemical analyses of extracts were made by following standard procedures. The broth microdilution method and the checkerboard methods were used to determine the antifungal activity and the combination of extracts with antifungals drugs. The methanol extract of seeds and the hexane extract of leaves exhibited the best significant antifungal activity with MIC values of 32 µg/mL. Furthermore, the combination of both extracts with nystatin and clotrimazole showed synergistic interactions with a 32-fold reduction in the MIC values of nystatin. Our findings indicate that Eriobotrya japonica extracts are a potential source of new antifungals that could be developed for use in the treatment of cryptococcosis. The anticryptococcal and antifungal activities potentiating activity of the studied extracts indicate their potential in the management of cryptococcosis. Further study should be considered to identify the bioactive principles against Cryptococcus neoformans.

Evaluation of a Cryptococcus capsular polysaccharide detection FungiXpert LFA (lateral flow assay) for the rapid diagnosis of Cryptococcosis.

The FungiXpert LFA was developed to diagnose fungal meningitis caused by Cryptococcus yeasts, by using serum or cerebrospinal fluid. It was compared to an existing lateral flow assay (LFA). The FungiXpert LFA performed well in qualitative and semi-quantitative tests.

Drug repositioning of benzimidazole anthelmintics in the treatment of cryptococcosis: a review

Drug repositioning of benzimidazole anthelmintics in the treatment of cryptococcosis: a review

Titan Cells and Yeast Forms of Cryptococcus neoformans and Cryptococcus gattii Are Recognized by GXMR-CAR

Cryptococcosis, a systemic mycosis that affects both the immunocompromised and immunocompetent, is caused by the inhalation of dehydrated yeasts or fungal spores of Cryptococcus gattii or Cryptococcus neoformans. The Cryptococcus spp. polysaccharide capsule is composed mainly of glucuronoxylomannan—GXM, its major virulence factor. The capsule thickness increases to more than 15 μm during titanization, favoring the pathogenesis of cryptococcosis. Previous studies demonstrated that cytotoxic T cells that had been bioengineered with GXM-targeting chimeric antigen receptor (GXMR-CAR) were able to recognize C. neoformans by promoting the control of titanization. GXMR-CAR, a second-generation CAR, contains a single-chain variable fragment that originates from a 18B7 clone: a human IgG4 hinge, followed by a human CD28 (transmembrane/cytoplasmic domains) and a CD3ς chain. In the current study, we redirected T cells to target distinct C. neoformans and C. gattii cell types by GXMR-CAR. Lentiviral particles carrying the GXMR-CAR sequence were used to transduce Jurkat cells, and these modified cells interacted with the GXM of the C. gattii R265 strain. Moreover, GXMR-CAR mediated the recognition of C. gattii and C. neoformans yeasts with both thin and thick polysaccharide capsules, and GXMR-CAR Jurkat cells interacted with titan cells sourced from both Cryptococcus spp. Thus, bioengineered cells using CAR can improve the treatment of cryptococcosis.

Progressive Cytopenia Developing during Treatment of Cryptococcosis in a Patient with HIV Infection and Bone Marrow Cryptococcal Infection.

Cytopenia is a common complication in patients with human immunodeficiency virus (HIV) infection. Identifying the cause is demanding because of the wide range of possible diagnoses. We herein report an HIV-infected patient with disseminated cryptococcosis involving multiple organs including the blood, brain, lungs, and bone marrow, who developed progressive pancytopenia after initiation of anti-fungal treatment with liposomal amphotericin-B (L-AMB) and flucytosine (5FC). The pancytopenia persisted despite early 5FC discontinuation. A bone marrow biopsy revealed cryptococcal infiltration and the blood examination findings recovered quickly after resuming L-AMB. Thus, this HIV-infected patient's pathological findings and clinical course suggested that the primary cause of the pancytopenia was bone marrow cryptococcosis.

Liposomal Ellagic Acid Alleviates Cyclophosphamide-Induced Toxicity and Eliminates the Systemic Cryptococcus neoformans Infection in Leukopenic Mice.

Cryptococcus neoformans infections rose sharply due to rapid increase in the numbers of immunocompromised individuals in recent years. Treatment of Cryptococcosis in immunocompromised persons is largely very challenging and hopeless. Hence, this study aimed to determine the activity of ellagic acid (EA) in the treatment of C. neoformans in cyclophosphamide injected leukopenic mice. A liposomal formulation of ellagic acid (Lip-EA) was prepared and characterized, and its antifungal activity was assessed in comparison to fluconazole (FLZ). The efficacy of the drug treatment was tested by assessing survival rate, fungal burden, and histological analysis in lung tissues. The safety of the drug formulations was tested by investigating hepatic, renal function, and antioxidant levels. The results of the present work demonstrated that Lip-EA, not FLZ, effectively eliminated C. neoformans infection in the leukopenic mice. Mice treated with Lip-EA (40 mg/kg) showed 70% survival rate and highly reduced fungal burden in their lung tissues, whereas the mice treated with FLZ (40 mg/kg) had 20% survival rate and greater fungal load in their lungs. Noteworthy, Lip-EA treatment alleviated cyclophosphamide-induced toxicity and restored hepatic and renal function parameters. Moreover, Lip-EA treatment restored the levels of superoxide dismutase and reduced glutathione and catalase in the lung tissues. The effect of FLZ or EA or Lip-EA against C. neoformans infection was assessed by the histological analysis of lung tissues. Lip-EA effectively reduced influx of inflammatory cells, thickening of alveolar walls, congestion, and hemorrhage. The findings of the present study suggest that Lip-EA may prove to be a promising therapeutic formulation against C. neoformans in immunocompromised persons.