Marife Martinez

Significant differences in drug susceptibility among species in the Candida parapsilosis group

Candida parapsilosis family has 3 proposed species: C. parapsilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. C. parapsilosis sensu stricto had significantly higher caspofungin (CAS) and anidulafungin MICs than C. orthopsilosis or C. metapsilosis; C. metapsilosis was least susceptible to fluconazole. C. parapsilosis sensu stricto more frequently displayed (37%) paradoxical growth in CAS (P < or = 0.02). These species susceptibility differences could affect therapeutic choices.

Efficacy of Ravuconazole in Treatment of Systemic Murine Histoplasmosis

ABSTRACT Ravuconazole (RCZ) was evaluated for efficacy in comparison to fluconazole (FCZ) and itraconazole (ITZ) in murine models of disseminated histoplasmosis. All regimens tested prolonged survival (P < 0.05 to 0.0001). At equivalent doses of 50 mg/kg of body weight, RCZ and ITZ were equally effective and RCZ was more effective than FCZ (P = 0.02). Clearance of fungal burden from the livers and spleens of mice showed RCZ and ITZ at doses of 50 mg/kg to be efficacious but not curative. These data indicate that RCZ should be studied further.

Saccharomyces as a vaccine against systemic candidiasis.

We have shown heat-killed Saccharomyces (HKY) is a protective vaccine against aspergillosis and coccidioidomycosis. To test the hypothesis that the efficacy of HKY- induced protection may be due to the cross-reactive antigens in the cell walls of the different fungi, we studied the effect of HKY against systemic candidiasis. Male CD-1 mice were given different regimens of HKY subcutaneously prior to intravenous challenge with Candida albicans. Compared to PBS controls, the administration of HKY (6 × 107) 3, 4 or 6 times prolonged survival (all P < 0.05) and reduced fungal load in the kidney (all P < 0.05). An HKY dose of 1.2 × 108 given 4 times prolonged survival (P = 0.02), but showed dose-limiting toxicity. HKY given by an oral route, or by a subcutaneous route with alum as an adjuvant, did not improve survival. Overall, we found that HKY protects mice from infection by Candida albicans in a dose-and regimen-dependent manner. To understand the protection induced by HKY against different fungal species, additional studies of epitope mapping are warranted.

Efficacy of voriconazole in Japanese quail (Coturnix japonica) experimentally infected with Aspergillus fumigatus

Aspergillus fumigatus causes disease in birds. Our objectives were to determine pharmacokinetic parameters and evaluate efficacy of voriconazole (VCZ) in a novel experimental quail model. After a single oral VCZ dose of 20 or 40 mg/kg, plasma concentrations peaked 2 h postdose (5.8 and 6.9 μg/ml) and remained above 0.5 μg/ml for 4 and 12 h postdose, respectively. For the efficacy study, ten-day-old Japanese quail (n = 60) were infected intratracheally with A. fumigatus conidia. Daily oral VCZ at 20 or 40 mg/kg was initiated 24 h postinfection (PI); infected diluent-treated birds were given de-ionized water orally. Preassigned birds were euthanized on days 5 or 10 PI. VCZ at 40 mg/kg prolonged survival compared to 20 mg/kg or diluent-treatment (P < 0.05) and lungs from birds given VCZ at 40 mg/kg had fewer colony forming units (CFU) than diluent-treated (P = 0.03). At day 10 PI, birds treated with VCZ at 20 mg/kg had significantly fewer fungi in the lungs as demonstrated by methenamine silver stain (P = 0....

Aspergillus fumigatus Invasion Increases with Progressive Airway Ischemia

Despite the prevalence of Aspergillus-related disease in immune suppressed lung transplant patients, little is known of the host-pathogen interaction. Because of the mould’s angiotropic nature and because of its capacity to thrive in hypoxic conditions, we hypothesized that the degree of Aspergillus invasion would increase with progressive rejection-mediated ischemia of the allograft. To study this relationship, we utilized a novel orthotopic tracheal transplant model of Aspergillus infection, in which it was possible to assess the effects of tissue hypoxia and ischemia on airway infectivity. Laser Doppler flowmetry and FITC-lectin were used to determine blood perfusion, and a fiber optic microsensor was used to measure airway tissue oxygen tension. Fungal burden and depth of invasion were graded using histopathology. We demonstrated a high efficacy (80%) for producing a localized fungal tracheal infection with the majority of infection occurring at the donor-recipient anastomosis; Aspergillus was more invasive in allogeneic compared to syngeneic groups. During the study period, the overall kinetics of both non-infected and infected allografts was similar, demonstrating a progressive loss of perfusion and oxygenation, which reached a nadir by days 10-12 post-transplantation. The extent of Aspergillus invasion directly correlated with the degree of graft hypoxia and ischemia. Compared to the midtrachea, the donor-recipient anastomotic site exhibited lower perfusion and more invasive disease; a finding consistent with clinical experience. For the first time, we identify ischemia as a putative risk factor for Aspergillus invasion. Therapeutic approaches focused on preserving vascular health may play an important role in limiting Aspergillus infections.

Vaccination with mannan protects mice against systemic aspergillosis

Invasive aspergillosis is a major cause of mortality in immunocompromised patients and therapeutic options are often limited, thus a vaccine would be desirable. We presently studied acid-stable cell-wall mannan (α-1, 6-linked backbone highly branched with α-1, 2; α-1, 3; and β-1, 2-linked manno-oligomers) derived from C. albicans, with or without conjugation to bovine serum albumin (BSA), as a vaccine against systemic aspergillosis. Mice were vaccinated subcutaneously with mannan or mannan-BSA conjugate weekly 3 times, ending 2 weeks prior to infection with A. fumigatus conidia. Results showed that the protection induced by mannan is dose-dependent; 12 mg unconjugated mannan alone or > 0.3 mg mannan-BSA consistently enhanced survival (P < 0.05). Fungal burdens in brains and kidneys were reduced after > 0.3 mg of mannan-BSA (all P < 0.05). Mannan-induced protection was improved about 40-fold by conjugation of BSA to mannan. Mannan-BSA (500 kDa) was more protective than 40 kDa mannan-BSA. Mannan is a candidate for a cross-protective conjugate fungal vaccine.

Resistance of MBL gene-knockout mice to experimental systemic aspergillosis.

Mannose binding lectin (MBL) is a protein of the collectin family that appears important in resistance to invasive pulmonary aspergillosis. We assessed the role of MBL in experimental systemic aspergillosis. MBL-sufficient C57BL/6 (WT) mice and B6.129S4--Mb11(tm1Kata) Mb12(tm1Kata)/J MBL A and C gene-knockout (KO) mice were infected intravenously with different inocula of Aspergillus fumigatus conidia. WT and KO mice were dose-responsively susceptible. In no instance were the KO mice more susceptible than WT. At the highest inoculum, all WT and 90% of KO mice died on day 4 (P>0.05). Reduction of the inoculum to 5.5 x 10(6) conidia was lethal, but comparison showed KO mice less susceptible to lethal infection (P<0.015). At the lowest inoculum used, deaths of KO mice were delayed, but survival was not significantly different than WT (P>0.05). These results suggest MBL may play a deleterious role in systemic aspergillosis.

Comparative Efficacies of Lipid-Complexed Amphotericin B and Liposomal Amphotericin B against Coccidioidal Meningitis in Rabbits

ABSTRACT In separate previous studies, we have shown that lipid-complexed amphotericin B (Abelcet [ABLC]) and liposomal amphotericin B (AmBisome [AmBi]) are efficacious against coccidioidal meningitis in rabbits. Here, we compared ABLC and AmBi directly in a coccidioidal meningitis model. Male New Zealand White rabbits were infected with 5 × 104Coccidioides posadasii arthroconidia by direct cisternal puncture. Therapy with intravenous ABLC or AmBi at 7.5 or 15 mg/kg of body weight or sterile 5% dextrose water (D5W) began 5 days later. Clinical assessments were done daily; cerebrospinal fluid and blood samples were obtained on day 15 and upon euthanasia. Survivors to day 25 were euthanatized, the numbers of CFU in their tissues were determined, and histology analyses of the brains and spinal cords were done. Controls showed progressive disease, whereas animals treated with either dose of either drug showed few clinical signs of infection. All ABLC- or AmBi-treated rabbits survived, whereas eight of nine D5W-treated rabbits were euthanatized before day 25 (P < 0.0001). Numbers of CFU in the brains and spinal cords of ABLC- or AmBi-treated animals were 100- to 10,000-fold lower than those in the corresponding tissues of D5W-treated animals (P < 0.0006 to 0.0001). However, only two or fewer given a regimen of ABLC or AmBi were cured of infection in both tissues. Fewer ABLC-treated rabbits (four of eight treated with 7.5 mg/kg and five of eight treated with 15 mg/kg) than controls (nine of nine) had meningitis at any level of severity (P, 0.015 or 0.043 for animals treated with ABLC at 7.5 or 15 mg/kg, respectively). Although groups of rabbits treated with AmBi regimens did not have significantly fewer animals with meningitis than the control group (P > 0.05), ABLC and AmBi were not significantly different. In this model, intravenous ABLC and AmBi were similarly highly effective, with few clinical signs of infection, 100% survival, and significantly reduced fungal burdens among treated animals. There appeared to be little benefit in using the 15-mg/kg dosage of either formulation. There was no significant advantage of one drug over the other for this indication. Further studies are required to determine the lowest effective doses of these formulations.

Effects of Iron Chelators on the Formation and Development of Aspergillus fumigatus Biofilm

ABSTRACT Iron acquisition is crucial for the growth of Aspergillus fumigatus. A. fumigatus biofilm formation occurs in vitro and in vivo and is associated with physiological changes. In this study, we assessed the effects of Fe chelators on biofilm formation and development. Deferiprone (DFP), deferasirox (DFS), and deferoxamine (DFM) were tested for MIC against a reference isolate via a broth macrodilution method. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to DFP, DFM, DFP plus FeCl3, or FeCl3 alone. A preformed biofilm was exposed to DFP with or without FeCl3. The DFP and DFS MIC50 against planktonic A. fumigatus was 1,250 μM, and XTT gave the same result. DFM showed no planktonic inhibition at concentrations of ≤2,500 μM. By XTT testing, DFM concentrations of <1,250 μM had no effect, whereas 2,500 μM increased biofilms forming in A. fumigatus or preformed biofilms (P < 0.01). DFP at 156 to 2,500 μM inhibited biofilm formation (P < 0.01 to 0.001) in a dose-responsive manner. Biofilm formation with 625 μM DFP plus any concentration of FeCl3 was lower than that in the controls (P < 0.05 to 0.001). FeCl3 at ≥625 μM reversed the DFP inhibitory effect (P < 0.05 to 0.01), but the reversal was incomplete compared to the controls (P < 0.05 to 0.01). For preformed biofilms, DFP in the range of ≥625 to 1,250 μM was inhibitory compared to the controls (P < 0.01 to 0.001). FeCl3 at ≥625 μM overcame inhibition by 625 μM DFP (P < 0.001). FeCl3 alone at ≥156 μM stimulated biofilm formation (P < 0.05 to 0.001). Preformed A. fumigatus biofilm increased with 2,500 μM FeCl3 only (P < 0.05). In a strain survey, various susceptibilities of biofilms of A. fumigatus clinical isolates to DFP were noted. In conclusion, iron stimulates biofilm formation and preformed biofilms. Chelators can inhibit or enhance biofilms. Chelation may be a potential therapy for A. fumigatus, but we show here that chelators must be chosen carefully. Individual isolate susceptibility assessments may be needed.

Protection against experimental aspergillosis by heat-killed yeast is not antibody dependent

Previously we showed heat-killed yeast (HKY) of Saccharomyces cerevisiae administered as a vaccine are protective against systemic murine aspergillosis (and other mycoses) and that HKY induces antibody and cellular responses. To determine the role of antibodies in this protection, male antibody knockout mice (KO; strain B6.129S2-Igh-6 (tm1Cgn)/J) and C57BL/6 wild-type (WT) mice were vaccinated subcutaneously with 6 × 10(7) HKY or phosphate buffered saline (PBS) given three or four times. Mice were infected intravenously with 6 × 10(6) viable conidia of Aspergillus fumigatus 10AF and mortality tallied through 12 days post infection. HKY vaccination given four times proved protective in the prolongation of survival of WT and KO mice vs. the respective PBS-treated controls. In one study, survival was prolonged in vaccinated WT or KO mice (P < 0.0001). A second study confirmed these results (P < 0.0001). Additionally, a three-dose regimen of HKY was also effective, prolonging survival of WT or KO mice vs. controls (P = 0.0002); no difference was found when the effectiveness of three- or four-dose regimens was compared. No significant differences in survival were found between HKY-vaccinated WT and KO mice, nor were PBS-treated KO mice more susceptible to infection than PBS-treated WT mice. Similar results were noted in another study in which a higher infectious inoculum and a three-dose regimen were used. Overall, antibodies do not appear to play a significant role in HKY-induced prolongation of survival in systemic aspergillosis, nor do antibodies appear to play a role in the innate resistance of the mice to aspergillosis.