Meningitis Associated with Simultaneous Infection by Multiple Dengue Virus Serotypes in Children, Brazil

To determine the causes of viral meningitis, we analyzed 22 cerebrospinal fluid samples collected during the 2014–2015 dengue epidemics in Brazil. We identified 3 serotypes of dengue virus (DENV-1, -2, and -3), as well as co-infection with 2 or 3 serotypes. We also detected the Asian II genotype of DENV-2

Fluconazole Alters the Polysaccharide Capsule of <i>Cryptococcus gattii</i> and Leads to Distinct Behaviors in Murine Cryptococcosis

<div><p><i>Cryptococcus gattii</i> is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of <i>C. gattii</i> was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01<b>_F</b>, that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01_F were altered by fluconazole. L27/01<b>_F</b> presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01<b>_F</b> did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/01<b>_F</b> presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01<b>_F</b>. Our results suggest that stress to fluconazole alters the capsule of <i>C. gattii</i> and influences the clinical manifestations of cryptococcosis.</p></div

Reduced susceptibility to fluconazole leads to very low <i>CAP59</i> and <i>CAP64</i> gene expression and affects physical properties of the GXM capsule.

<p>Microscopic visualization with India ink stain of L27/01 (<b>A</b>) and L27/01<b>_F</b> strains (<b>B</b>). Capsule size of L27/01 and L27/01<b>_F</b> strains (<b>C</b>). SEM analysis of L27/01 (<b>D</b>) and L27/01<b>_F</b> (<b>E</b>) strains. Zeta potential of capsular and secreted PS of cells (<b>F</b>). Cell suspensions were analyzed with an AXIOPLAN (Carl Zeiss) fluorescence microscope. Images were processed using ImageJ. Staining of L27/01 (<b>G</b>) and L27/01<b>_F</b> (<b>H</b>) strains with mAb 18B7 showing differences in epitope presentation in the PS capsule between strains. Ratio of gene expression of <i>CAP59</i>, <i>CAP64</i> and <i>UXS1</i> to actin (<b>I</b>). Size distribution of PS fibers from capsular (<b>J</b> and <b>K</b>) and exo-PS samples (<b>L</b> and <b>M</b>) of L27/01 and L27/01<b>_F</b> strains, respectively. Bar = 10 µm (A) and (D). Bar = 5 µm (B), (E), (G) and (H). *P<0.05 was considered to be significant.</p

L27/01 alters the clinical manifestations of cryptococcosis.

<p>Assessment of behavioral performance of not infected (NI) mice and those infected with L27/01 (▪) or L27/01<b>_F</b> (▴) strains in five distinct functional categories of the SHIRPA battery (n = 6) (<b>A</b>–<b>E</b>). Mice were monitored daily for: muscle tone and strength (<b>A</b>); motor behavior (<b>B</b>); neuropsychiatric state (<b>C</b>) autonomous function (<b>D</b>); reflex and sensory function (<b>E</b>) (n = 6). The results are presented mean ± SEM of eight animals in each group. Scores of the infected groups were compared with the uninfected group by the Wilcoxon matched test. *The vertical dotted line indicates a significant difference from the appointed day. P<0.05 is considered to be significant.</p

Synopsis of the methodology.

<p>Fluconazole-resistant strain selection (<b>A–F</b>). After determining the MIC of fluconazole on SDA, an average of five colonies obtained from the highest fluconazole concentration were selected for culture on SDA plates supplemented with this drug. L27/01 (<b>A</b>) was the strain able to grow at the highest fluconazole concentration (<b>B</b>) and was chosen for culture on SDA plates supplemented with this drug. This strain was cultured in solid medium with increasing concentrations (<b>B–C</b>) of fluconazole until growth ceased at 100 µg/mL (<b>D</b>). To verify the maintenance of resistance to fluconazole and cross-resistance between this drug and amphotericin B, the selected strain was cultured in SDA without drug every 48 h 170 times (<b>E</b>), and the MIC test was performed by microdilution every 5 subcultures (<b>F</b>). Colonies grown at 95 µg/mL were maintained in this concentration, and the strain was named “L27/01<b>_F</b>” (<b>G</b>). L27/01 strain grew in the absence of drug (<b>H</b>). The genetic similarity between L27/01 and L27/01<b>_F</b> strains was evaluated by randomly amplified polymorphic DNA (RAPD)-PCR, PFGE and ITS Sequencing. <i>CAP59, CAP64, ARF-1, ERG11, UXS-1</i> levels by real-time PCR were evaluated. Lipid evaluation was performed to compare the ergosterol content of L27/01 and L27/01<b>_F</b> cell membranes. Also, Urease and Laccase activities of L27/01 and L27/01<b>_F</b> strains were determined (<b>I</b>). Evaluation of murine cryptococcosis after inoculation with L27/01 or L27/01<b>_F</b> strain: survival curve (<b>J</b>). Investigation of whether fluconazole affects the polysaccharide (PS) capsule (<b>K</b>). The phagocytosis assay was performed to assess the influence of PS capsules from L27/01 and L27/01<b>_F</b> strains on phagocytosis and intracellular proliferation rate (IPR) in murine peritoneal macrophages from C57BL/6 mice (<b>L</b>). Cryptococcal cell dissemination, immune response and behavioral alterations (<b>M</b>). SDA: Sabouraud Dextrose Agar. MIC: Minimum inhibitory concentration. PFGE: Pulsed field gel electrophoresis. CFU: Colony forming units. BALF: bronchoalveolar lavage fluid. MPO: Myeloperoxidase activity. i.t.: intratracheal infection. PS: polysaccharide.</p

The L27/01_F strain is more readily internalized by macrophages, but is not able to proliferate as well as the L27/01 strain.

<p>The phagocytosis assay was performed <i>in vivo</i> by counting internalized yeast cells in BALF cells, and <i>in vitro</i> with murine peritoneal macrophages infected with <i>C. gattii</i>. The influence of L27/01 and L27/01<b>_F</b> polysaccharide capsules (L27/01 PS and L27/01<b>_F</b> PS) on intracellular proliferation rate (IPR), reactive oxygen species, superoxide-dismutase (SOD) and peroxidase (PER) activities was also evaluated. White bars refer to macrophages (control), black bars to L27/01, grey bars to L27/01<b>_F</b> and beige bars to PS alone without yeasts. Phagocytic index (<b>A</b>). IPR (<b>B</b>). Reactive oxygen species expressed in arbitrary units of fluorescence (AU) (<b>C</b>). SOD (U mg⁻¹ protein) (<b>D</b>) and PER (nmol/min mg⁻¹ protein) (<b>E</b>) activities after phagocytosis. The data are presented as the mean ± S.E.M. of two independent experiments in triplicate. *P<0.05. Symbols indicate absence (–) of PS in the phagocytosis assay.</p

Reduced susceptibility to fluconazole impairs <i>C. gattii</i> migration to the CNS.

<p>Lungs (<b>A</b>), brain (<b>B</b>), and Bronchoalveolar lavage fluid (BALF) (<b>C</b>) were removed (n = 8), homogenized, diluted and plated onto Sabouraud dextrose agar for measurement of fungal burden 0.5, 1, 7, 15, and 30 days post-infection with 10⁶ cells of L27/01 or L27/01<b>_F</b> strains. ND: non-detected. Black bars refer to L27/01 and grey bars refer to L27/01<b>_F</b>. *Data represent the mean of two independent experiments in triplicate. P<0.05 was considered to be significant. (<b>D–H</b>) Histopathological sections stained with H&E of lung and brain 15 days post-inoculation. Lungs of control mice (<b>D</b>). Lungs of mice inoculated with L27/01 strain showing mild peribronchiolar and moderate perivascular neutrophilic and histiocytic inflammatory infiltrate associated with numerous yeast cells in the peribronchiolar and alveolar lumen (<b>E</b>). Lungs of mice inoculated with L27/01<b>_F</b> strain, with intense diffuse inflammatory infiltrate, neutrophils and macrophages around few yeast cells (head arrow) (<b>F</b>). Bar = 40 µm. Hippocampi of mice inoculated with L27/01 strain showed cryptococcoma (arrow), neuronal degeneration and death (head arrow). <i>Cryptococcus gattii</i> yeasts in cryptococcoma were stained with Groccot’s stain (in detail) (<b>G</b>). Hippocampi of mice inoculated with L27/01<b>_F</b> strain showed unaltered structure (<b>H</b>). Bar = 20 µm.</p

L27/01 developed reduced susceptibility to fluconazole with overexpression of the <i>MDR1</i> gene, but the parental strain is more virulent than L27/01_F in mice.

<p>Pulsed field gel electrophoresis (PFGE) patterns of L27/01_F and L27/01 and strains on 1% PFGE certified agarose (lanes 2 and 3, respectively). Lane 1 show PFGE Size Marker (SM), 0.225–2.2 Mb <i>S. cerevisiae</i> chromosomal DNA <i>Saccharomyces cerevisiae</i> used as size standard (<b>A</b>). <i>MDR1</i> gene expression in L27/01 and L27/01_F strains (<b>B</b>). Ergosterol levels of L27/01 and L27/01<b>_F</b> strains (<b>C</b>). Urease and Laccase activities of L27/01 and L27/01<b>_F</b> strains: one enzymatic unit was calculated per 10⁵ and 10⁸ cells of <i>C. gattii</i> for urease and laccase activity, respectively, after 24 hours (<b>D</b>). Eight mice per group were inoculated by the intratracheal route with 10⁶ cells of L27/01 or L27/01<b>_F</b>. Animals administered with PBS represent the non-infected (control) group. Animals were monitored daily. Survival curve (<b>E</b>). Weight variation (Score) (<b>F</b>). Each 1 (one) gram received or lost corresponds to 10 points added or subtracted, respectively. *The vertical dotted line indicates a significant difference from the appointed day. CFU/g of lungs (<b>G</b>) from animals infected with 1×10⁶ cells of L27/01 or L27/01<b>_F</b> strains treated i.p. with fluconazole at 10 mg/kg/day (n = 6). NI: not infected group. NT: non treated group. Black bars refer to L27/01 (▪) and grey bars refer to L27/01<b>_F</b> (▴). Data represent the mean of three independent experiments in triplicate. *P<0.05 was considered to be significant.</p

L27/01_F increases recruitment of host cells to the lungs, but decreases cytokine production.

<p>L27/01 and L27/01<b>_F</b> strains elicit different inflammatory response in C57/BL6 mice. Mice (n = 8) were inoculated i.t. with 10⁶ yeast cells of L27/01 or L27/01<b>_F</b> strains. After 1, 7, 15 or 30 days post infection, the mice were euthanized and mononuclear leukocytes (<b>A</b>) and neutrophils (<b>B</b>) in bronchoalveolar lavage fluid (BALF) were quantified. Neutrophilic infiltrate in the lungs was also determined as MPO activity and expressed as relative number of neutrophils (<b>C</b>). Levels of CXCL1/KC (<b>D</b>), IFN-γ (<b>E</b>), TNF-α (<b>F</b>) and IL-10 (<b>G</b>) are expressed in pg per 100 mg of lung. White bars refer to not infected (NI) animals, black bars to animals infected with the L27/01 strain, and grey bars to animals infected with the L27/01<b>_F</b> strain. The results are presented as the mean ± SEM. Each experiment was performed in triplicate. *P<0.05 was considered to be significant.</p