Clinical Isolates Of Cryptococcus Research Articles

GXMR-CAR containing distinct GXM-specific single-chain variable fragment (scFv) mediated the cell activation against Cryptococcus spp. And had difference in the strength of tonic signaling

ABSTRACTCryptococcus spp. has a polysaccharide capsule composed of glucuronoxylomannan-GXM, a major virulence factor that can prevent the recognition of fungi by immune cells. Chimeric Antigen Receptor (CAR) redirects T cells to target Cryptococcus spp. as previously demonstrated by a CAR specific to GXM, GXMR-CAR. The current study evaluated the strength of the signal transduction triggered by GXMR-CAR, composed of a distinct antigen-binding domain sourced from a single-chain variable fragment (scFv). GXM-specific scFv derived from mAbs 2H1 and 18B7, 2H1-GXMR-CAR and 18B7-GXMR-CAR, respectively, were designed to express CD8 molecule as hinge/transmembrane, and the costimulatory molecule CD137 (4-1BB) coupled to CD3ζ. The 2H1-GXMR-CAR or 18B7-GXMR-CAR Jurkat cells recognized soluble GXM from C. gattii and C. neoformans, and the levels of IL-2 released by the modified cells did not differ between the GXMR-CAR constructs after exposure to Cryptococcus spp. 18B7-GXMR-CAR triggered tonic signaling was more pronounced in modified Jurkat cells, and a protein kinase inhibitor of the Src family (dasatinib) significantly reduced GXMR-CAR tonic signaling and inhibited cell activation against ligands. 18B7 scFv showed a structural modification of the variable heavy (VH) chain that clarified the difference in the strength of tonic signaling and the level of cell activation between 2H1-GXMR-CAR and 18B7-GXMR-CAR. GXMR-CAR constructs induced T-cell activation against clinical isolates of Cryptococcus spp. and serum from patients with cryptococcosis induced high levels of IL-2, mainly in cells modified with 18B7-GXMR-CAR. Thus, 18B7-GXMR-CAR and 2H1-GXMR-CAR mediated T cell activation against Cryptococcus spp. and 18B7 and 2H1 scFv influenced the strength of tonic signaling.

Determining Potential Link between Environmental and Clinical Isolates of Cryptococcus neoformans/Cryptococcus gattii Species Complexes Using Phenotypic and Genotypic Characterisation

Opportunistic infections due to Cryptococcus neoformans and C. gattii species complexes continue to rise unabated among HIV/AIDS patients, despite improved antifungal therapies. Here, we collected a total of 20 environmental and 25 presumptive clinical cryptococcal isolates from cerebrospinal fluid (CSF) samples of 175 patients enrolled in an ongoing clinical trial Ambition 1 Project (Botswana-Harvard Partnership). Identity confirmation of the isolates was done using MALDI-TOF MS and PCR. We describe the diversity of the isolates by PCR fingerprinting and sequencing (Oxford Nanopore Technology) of the intergenic spacer region. Mating types of the isolates were determined by amplification of the MAT locus. We report an unusual prevalence of 42.1% of C. neoformans x C. deneoformans hybrids Serotype AD (n = 16), followed by 39.5% of C. neoformans Serotype A (n = 15), 5.3% of C. deneoformans, Serotype D (n = 2), 7.9% of C. gattii (n = 3), and 5.3% of C. tetragattii (n = 2) in 38 representative isolates that have been characterized. Mating type-specific PCR performed on 38 representative environmental and clinical isolates revealed that 16 (42.1%) were MAT a/ MATα hybrids, 17 (44.7%) were MATα, and five (13.2%) possessed MAT a mating type. We used conventional and NGS platforms to demonstrate a potential link between environmental and clinical isolates and lay a foundation to further describe mating patterns/history in Botswana.

A Study of the Disruptive Effect of the Acetate Fraction of Punica granatum Extract on Cryptococcus Biofilms.

Cryptococcosis, caused by yeasts of the genus Cryptococcus, is an infectious disease with a worldwide distribution. Cryptococcus neoformans and Cryptococcus gattii are the species that commonly cause this disease in humans; however, infections caused by Cryptococcus laurentii, especially in immunocompromised patients, are increasingly being reported. Owing to the increase in the resistance of fungi to antifungals, and a lack of treatment options, it is important to seek new therapeutic alternatives such as natural products. Among these are plant species such as Punica granatum, which is used in folk medicine to treat various diseases. This study aimed to evaluate the activity of the acetate fraction of P. granatum leaf extract against environmental and clinical isolates of Cryptococcus. Three environmental isolates of C. laurentii, PMN, PMA, and PJL II, isolated from soils of different municipalities in the state of Maranhão, a clinical isolate, C. gattii, from a patient with neurocryptococcosis, and a standard strain of C. gattii (ATCC 32068) were used. The minimum and fractional inhibitory concentrations (MIC and FIC, respectively) and time-kill curve of the extract and fluconazole were determined to assess the susceptibility profile of the fungal isolates. Larvae of Tenebrio molitor were infected with Cryptococcus strains, and the effects of acetate fraction of P. granatum extract and fluconazole on the survival and fungal burden were determined. The extract activity was tested against pre-formed biofilms. The acetate fraction of P. granatum extract showed promising antifungal activity against all the species of Cryptococcus evaluated in this study, with an MIC value lower than that of fluconazole. The indices obtained in the FIC test indicated that the antimicrobial effect of the combination of the extract and antifungal was indifferent for 80% of the isolates. The P. granatum acetate fraction reduced the pre-formed biofilm of some isolates, showing better activity than fluconazole, which is consistent with results from fluorescence microscopy. This is the first study on the use of P. granatum and its ability to inhibit Cryptococcus biofilms; therefore, further studies and tests are needed to investigate the components and mechanism of action of P. granatum against cryptococcosis agents.

Cryptococcus neoformans and C. gattii isolates from both HIV-infected and uninfected patients: antifungal susceptibility and outcome of cryptococcal disease.

ABSTRACTOne of the factors causing treatment failure in cryptococcosis is the resistance of Cryptococcus spp. to antifungal drugs, which has motivated the susceptibility assessment of isolates from patients with cryptococcosis, different clinical conditions and infections outcomes. Clinical isolates of Cryptococcus spp. from three different groups of patients were studied in the present investigation: 19 HIV-positive patients with relapsing and/or refractory meningitis (Group 1), 30 HIV-positive patients who experienced a single and limited episode of cryptococcosis (Group 2), and 19 HIV-negative patients with cryptococcosis (Group 3). Eighty C. neoformans var. grubii isolates and 7 C. gattii isolates were studied. The minimum inhibitory concentration (MIC) of amphotericin B, azole drugs and flucytosine was determined for Cryptococcus spp. by broth microdilution test and E-test. The MIC50 and MIC90 were 0.25 and 0.50 µg/mL for amphotericin B, 4.0 and 8.0 µg /mL for fluconazole, 0.06 and 0.25 µg/mL for itraconazole, 0.25 and 0.50 µg/mL for voriconazole, and 8.0 and 16.0 µg/mL for flucytosine, respectively. Amphotericin B and itraconazole showed higher MICs for C. neoformans var. grubii and C. gattii, respectively. The MICs of fluconazole and itraconazole obtained with the E-test were higher than those obtained with broth microdilution. Isolates from non-HIV coinfected were less sensitive to the azoles. There was no difference in the susceptibility of C. neoformans var. grubii isolates from patients with a favorable or unfavorable outcome or along the episodes of relapsing and/or refractory meningitis.

Molecular typing of clinical isolates of Cryptococcus neoformans/Cryptococcus gattii species complex from Northeast Mexico.

Cryptococcosis is caused by members of the Cryptococcus neoformans/Cryptococcus gattii species complex. Based on molecular identification, these two species have been further differentiated into molecular types. The aim of this work was to characterize clinical cryptococcal isolates recovered from six hospitals in Northeast Mexico from 1995 to 2011. One hundred and sixty-six isolates, which were characterized by biochemical tests and in vitro susceptibility to amphotericin B, fluconazole, and voriconazole, and M13 PCR fingerprinting, were included in this study. Utilizing phenotypic tests, 153 isolates (92.16 %) were identified as C. neoformans and 13 (7.83 %) as C. gattii. All isolates were susceptible to all antifungals tested. Employing M13 PCR fingerprinting, eight molecular types were detected. VNI was the most common genotype (124 cases; 74.6 %), followed by VNII (15 cases; 9 %), VNIII (8 cases; 4.8 %), VNIV (6 cases; 3.6 %), VGI (6 cases; 3.6 %), VGII (3 cases; 1.8 %), and VGIII and VGIV (2 cases, 1.2 % each). We confirm the presence of C. gattii in clinical isolates in Northeast Mexico, and a high clonal diversity in the studied strains of C. neoformans/C. gattii species complex.

Global Trends in the Antifungal Susceptibility of Cryptococcus neoformans (1990 to 2004)

The antifungal susceptibilities of 1,811 clinical isolates of Cryptococcus neoformans obtained from 100 laboratories in 5 geographic regions worldwide between 1990 and 2004 were determined. The MICs of amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole, and ravuconazole were determined by the National Committee for Clinical Laboratory Standards broth microdilution method. Isolates were submitted to a central reference laboratory (University of Iowa) from study centers in Africa (5 centers, 395 isolates), Europe (14 centers, 102 isolates), Latin America (14 centers, 82 isolates), the Pacific region (7 centers, 50 isolates), and North America (60 centers, 1,182 isolates). Resistance to amphotericin B, flucytosine, and fluconazole was < or = 1% overall. Susceptibility to flucytosine (MIC, < or = 4 microg/ml) ranged from 35% in North America to 68% in Latin America. Similarly, only 75% of isolates from North America were susceptible to fluconazole (MIC, < or = 8 microg/ml) compared to 94 to 100% in the other regions. Isolates remained highly susceptible to amphotericin B (99% susceptibility at a MIC of < or = 1 microg/ml) over the entire 15-year period. Susceptibility to flucytosine (MIC, < or = 4 microg/ml) increased from 34% in 1990 to 1994 to 66% in 2000 to 2004. Susceptibility to fluconazole (MIC, < or = 8 microg/ml) increased from 72% in 1990 to 1994 to 96% in 2000 to 2004. Voriconazole, posaconazole, and ravuconazole all were very active (99% of isolates susceptible at MIC of < or = 1 microg/ml) against this geographically diverse collection of isolates. We conclude that in vitro resistance to antifungal agents used in the treatment of cryptococcosis remains uncommon among isolates of C. neoformans from five broad geographic regions and has not increased over a 15-year period.