Genotyping Analysis of Cryptococcus deuterogattii and Correlation with Virulence Factors and Antifungal Susceptibility by the Clinical and Laboratory Standards Institute and the European Committee on Antifungal Susceptibility Testing Methods.

Data about the relationship between their molecular types, virulence factors, clinical presentation, antifungal susceptibility profile, and outcome are still limited for Cryptococcus deuterogattii. This study aimed to evaluate the molecular and phenotypic characteristics of 24 C. deuterogattii isolates from the southeast region of Brazil. The molecular characterization was performed by multilocus sequence typing (MLST). The antifungal susceptibility profile was obtained according to CLSI-M27-A3 and EUCAST-EDef 7.1 methods. The virulence factors were evaluated using classic techniques. The isolates were divided into four populations. The molecular analysis suggests recombinant events in most of the groups evaluated. Resistance and susceptibility dose-dependent to fluconazole were evidenced in four isolates (16%) by EUCAST and in four isolates (16%) by CLSI methods. The agreement at ±two dilutions for both methods was 100% for itraconazole, ketoconazole, and voriconazole, 96% for amphotericin B, and 92% for fluconazole. Significant differences in virulence factor expression and antifungal susceptibility to itraconazole and amphotericin B were found. The mixed infection could be suggested by the presence of variable sequence types, differences in virulence factor production, and decreased antifungal susceptibility in two isolates from the same patient. The data presented herein corroborate previous reports about the molecular diversity of C. deuterogattii around the world.

Alternative use of phage display: phage M13 can remain viable in the intestines of poultry without causing damage.

Phage display (PD) is a tool for developing new molecules to control pathogens. Peptides selected by PD are commonly synthesised and tested, but the use of phage M13 displaying the selected peptides as a direct biding in the intestinal tract has not yet been tested. This study evaluated whether phage M13 can remain viable in the chicken gastrointestinal tract and whether it causes injury or humoral immune response. We inoculated phage M13 or E. coli ER2738 (ECR) infected with M13 into birds at different ages. We found the virus in faeces at 5 or 13 days after inoculation, just when it infected the ECR. The presence of phage M13 or ECR did not result in gut injuries and had no impacts on weight gain and bird health. Furthermore, the levels of IgY were similar in all treatments, which indicates that the virus can be used in chicken until 42 days without being recognised by the immune system. This work provides a scientific basis for the use of PD as a tool in numerous applications to control different pathogens.

Fatal Case of Fungemia by Wickerhamomyces anomalus in a Pediatric Patient Diagnosed in a Teaching Hospital from Brazil.

In recent decades, emerging fungal infections have changed the clinical mycology scenario as a consequence of the advances in medical diagnostics and therapeutic procedures, long hospitalization times, and the growing number of individuals with debilitating chronic diseases and impaired immune systems. This report presents a 19 months old Brazilian female patient who developed a severe fungal sepsis by an uncommon yeast. She was admitted at the intensive care unit with severe pneumonia, bronchopulmonary dysplasia, and weight-for-age z score of less than -2. She remained more than 30 days in the intensive care unit where she had a femoral venous catheter placement, enteral nutrition, broad-spectrum antibiotic therapy, and prophylaxis with fluconazole. Moreover, pericardiocentesis was performed due to cardiac tamponade. She had a previous history of prematurity, cardiac surgery due to patent ductus arteriosus, and a long period of hospital stay. Despite the antifungal prophylaxis, two yeast isolates were recovered from blood and then identified by classical mycological methods and internal transcribed spacer (ITS) sequencing as Wickerhamomyces anomalus. Both isolates exhibited susceptibility to amphotericin B, ketoconazole, itraconazole, voriconazole, and fluconazole. Her clinical state worsened, presenting anasarca, epistaxis, and hemorrhagic suffusions in the mouth, sclera, oliguria, and bradycardia. Two days after the first positive culture, she presented a gradual reduction of the white blood cells count, with severe leukopenia and neutropenia. She died five days after.

High genetic variability of clinical and environmental Cryptococcus gattii isolates from Brazil.

Among Cryptococcus gattii genotypes, VGII has gained pivotal relevance in epidemiological, clinical and genetic contexts due to its association with several outbreaks in temperate regions and due to the high variability of this genotype. The aim of this study was to compare 25 isolates of C. gattii from the Southeast region of Brazil with previously described isolates from other regions of the country and around the world. Among the 25 isolates, 24 were VGII and one was VGI. All of them were newly identified. Three new allele types (AT) (AT47 for the URA5 locus, AT56 for the LAC1 locus, and AT96 for the IGS1 region) were also described. Compared with other Brazilian isolates, those from the Southeast region presented the greatest haplotype diversity. In general, the regions presented different sequence types (STs), and only nine STs were found in more than one location. GoeBURST analysis showed two large groups among the Brazilian isolates. The largest group consists of 59 STs predominantly from the North and Northeast regions; the other large group includes 57 STs from the Southeast and Midwest regions. In a global context the South American isolates presented the highest genetic diversity (STs = 145, haplotype diversity (Hd) = 0.999 and π = 0.00464), while the African populations showed the lowest genetic diversity (STs = 3, Hd = 0.667 and π = 0.00225). These results confirm that the Brazilian C. gattii VGII population is highly diverse and reinforce the hypothesis of dispersion of this genotype from South America.

Synergic effect of simvastatin in combination with amphotericin B against environmental strains of Cryptococcus neoformans from northeastern Brazil: a prospective experimental study.

BACKGROUND: Statins are used as cholesterol-lowering drugs and may also have direct antimicrobial effects. OBJECTIVE: To evaluate synergic interactions between simvastatin and both amphotericin B and fluconazole, against environmental strains of Cryptococcus neoformans isolated from captive birds' droppings. DESIGNAND SETTING: Experimental study conducted at Federal University of Piauí, Parnaíba, in collaboration with Federal University of Triângulo Mineiro, Uberaba, Brazil. METHODS: Statin susceptibility tests of Cryptococcus neoformans samples were performed as prescribed in standards. Interactions of simvastatin with amphotericin and fluconazole were evaluated using the checkerboard microdilution method. Presence of these interactions was quantitatively detected through determining the fractional inhibitory concentration index (FICI). RESULTS: Isolates of Cryptococcus neoformans were obtained from 30 of the 206 samples of dry bird excreta (14.5%) that were collected from pet shops and houses. Ten isolates were selected for susceptibility tests. All of them were susceptible to amphotericin and fluconazole. All presented minimum inhibitory concentration (MIC) > 128 µg/ml and, thus, were resistant in vitro to simvastatin. An in vitro synergic effect was shown through combined testing of amphotericin B and simvastatin, such that six isolates (60%) presented FICI < 0.500. Two isolates showed considerable reductions in MIC, from 1 µg/ml to 0.250 µg/ml. No synergic effect was observed through combining fluconazole and simvastatin. CONCLUSION: These results demonstrate that simvastatin should be considered to be a therapeutic alternative, capable of potentiating the action of amphotericin B. However, further studies are necessary to clarify the real effect of simvastatin as an antifungal agent.

Synergic effect of simvastatin in combination with amphotericin B against environmental strains of Cryptococcus neoformans from northeastern Brazil: a prospective experimental study

BACKGROUND: Statins are used as cholesterol-lowering drugs and may also have direct antimicrobial effects. OBJECTIVE: To evaluate synergic interactions between simvastatin and both amphotericin B and fluconazole, against environmental strains of Cryptococcus neoformans isolated from captive birds' droppings. DESIGNAND SETTING: Experimental study conducted at Federal University of Piauí, Parnaíba, in collaboration with Federal University of Triângulo Mineiro, Uberaba, Brazil. METHODS: Statin susceptibility tests of Cryptococcus neoformans samples were performed as prescribed in standards. Interactions of simvastatin with amphotericin and fluconazole were evaluated using the checkerboard microdilution method. Presence of these interactions was quantitatively detected through determining the fractional inhibitory concentration index (FICI). RESULTS: Isolates of Cryptococcus neoformans were obtained from 30 of the 206 samples of dry bird excreta (14.5%) that were collected from pet shops and houses. Ten isolates were selected for susceptibility tests. All of them were susceptible to amphotericin and fluconazole. All presented minimum inhibitory concentration (MIC) > 128 µg/ml and, thus, were resistant in vitro to simvastatin. An in vitro synergic effect was shown through combined testing of amphotericin B and simvastatin, such that six isolates (60%) presented FICI < 0.500. Two isolates showed considerable reductions in MIC, from 1 µg/ml to 0.250 µg/ml. No synergic effect was observed through combining fluconazole and simvastatin. CONCLUSION: These results demonstrate that simvastatin should be considered to be a therapeutic alternative, capable of potentiating the action of amphotericin B. However, further studies are necessary to clarify the real effect of simvastatin as an antifungal agent.

Genotypic analysis of clinical and environmental Cryptococcus neoformans isolates from Brazil reveals the presence of VNB isolates and a correlation with biological factors.

Cryptococcal infections are mainly caused by members of the Cryptococcus neoformans species complex (molecular types VNI, VNII, VNB, VNIV and the AD hybrid VNIII). PCR of the mating type loci and MLST typing using the ISHAM-MLST consensus scheme were used to evaluate the genetic relationship of 102 (63 clinical and 39 environmental) C. neoformans isolates from Uberaba, Brazil and to correlate the obtained genotypes with clinical, antifungal susceptibility and virulence factor data. All isolates were mating type alpha. MLST identified 12 known and five new sequence types (ST). Fourteen STs were identified within the VNI isolates, with ST93 (57/102, 56%) and ST77 (19/102, 19%) being the most prevalent. From the nine VNII isolates previously identify by URA5-RFLP only four (ST40) were confirmed by MLST. The remaining five grouped within the VNB clade in the phylogenetic analysis corresponding to the sequence type ST504. Other two environmental isolates also grouped within VNB clade with the new sequence type ST527. The four VNII/ST40 isolates were isolated from CSF. The two VNIV sequence types (ST11 and ST160) were isolated from blood cultures. Two of six patients evaluated with more than one isolates had mixed infections. Amongst the VNI isolates 4 populations were identified, which showed differences in their susceptibility profiles, clinical outcome and virulence factors. These results reinforce that ST93 is the most prevalent ST in HIV-infected patients in the Southeastern region of Brazil. The finding of the VNB molecular type amongst environmental Brazilian isolates highlights that this genotype is not restricted to the African continent.

Phylogenetic analysis of phenotypically characterized Cryptococcus laurentii isolates reveals high frequency of cryptic species.

BACKGROUND: Although Cryptococcus laurentii has been considered saprophytic and its taxonomy is still being described, several cases of human infections have already reported. This study aimed to evaluate molecular aspects of C. laurentii isolates from Brazil, Botswana, Canada, and the United States. METHODS: In this study, 100 phenotypically identified C. laurentii isolates were evaluated by sequencing the 18S nuclear ribosomal small subunit rRNA gene (18S-SSU), D1/D2 region of 28S nuclear ribosomal large subunit rRNA gene (28S-LSU), and the internal transcribed spacer (ITS) of the ribosomal region. RESULTS: BLAST searches using 550-bp, 650-bp, and 550-bp sequenced amplicons obtained from the 18S-SSU, 28S-LSU, and the ITS region led to the identification of 75 C. laurentii strains that shared 99-100% identity with C. laurentii CBS 139. A total of nine isolates shared 99% identity with both Bullera sp. VY-68 and C. laurentii RY1. One isolate shared 99% identity with Cryptococcus rajasthanensis CBS 10406, and eight isolates shared 100% identity with Cryptococcus sp. APSS 862 according to the 28S-LSU and ITS regions and designated as Cryptococcus aspenensis sp. nov. (CBS 13867). While 16 isolates shared 99% identity with Cryptococcus flavescens CBS 942 according to the 18S-SSU sequence, only six were confirmed using the 28S-LSU and ITS region sequences. The remaining 10 shared 99% identity with Cryptococcus terrestris CBS 10810, which was recently described in Brazil. Through concatenated sequence analyses, seven sequence types in C. laurentii, three in C. flavescens, one in C. terrestris, and one in the C. aspenensis sp. nov. were identified. CONCLUSIONS: Sequencing permitted the characterization of 75% of the environmental C. laurentii isolates from different geographical areas and the identification of seven haplotypes of this species. Among sequenced regions, the increased variability of the ITS region in comparison to the 18S-SSU and 28S-LSU regions reinforces its applicability as a DNA barcode.