Improved detection of Candida albicans by PCR in blood of neutropenic mice with systemic candidiasis.

A PCR using primers aimed at the multicopy gene coding for the small subunit rRNA and resulting in the synthesis of a 180-bp fragment was evaluated for its use in diagnosing invasive candidiasis in comparison with blood culture. With the use of a C. albicans-specific probe, +/- 10 to 15 C. albicans cells are detected in 100 microliters of whole blood by Southern analysis. A DNase pretreatment was critical in the purification process of yeast DNA from whole blood. Omission of the DNase pretreatment decreased assay sensitivity 10-fold. PCR analysis of blood specimens collected from mice with invasive candidiasis is more sensitive than blood culture (100 versus 67%, respectively) at 72 h after intravenous (i.v.) inoculation with C. albicans. Furthermore, the intensity of the hybridization signals increased with the progression of infection. In contrast, multiple blood samples from gastrointestinally colonized mice were all negative by PCR, indicating that the PCR assay is also specific and may, therefore, make a positive contribution to the detection and follow-up of invasive candidiasis.     

Treatment of a murine model of systemic candidiasis with liposomal amphotericin B bearing antibody to Candida albicans

Survival of mice infected with an intravenous injection of Candida albicans was observed in a short-term (21-day) survival study. Concentration of C. albicans in the kidneys, liver, and spleen was determined at various times. The effects of treatment with the commercial formulation of amphotericin B (fAMB), liposomal amphotericin B (LAMB), and liposomal amphotericin B bearing external antibody specific for C. albicans (LAMB-Ab) were compared. In single intravenous treatment dosages of 0.6 mg of amphotericin B/kg, the liposomal forms of the drug (LAMB and LAMB-Ab) enhanced the percentage survival and mean survival time of mice in comparison with those treated with the unencapsulated antifungal compound, fAMB (p less than 0.03 and p less than 0.001, respectively). LAMB-Ab, at this dosage, produced an increase in the survival (p less than 0.007) of mice over that produced by LAMB. LAMB-Ab treatment caused a greater than 3-fold increase over fAMB. The percentage of LAMB-Ab-treated mice which survived for 21 days was almost double that of the LAMB-treated mice. The increase in survival following this treatment did not, however, lead to the eradication of C. albicans in all mice which survived to the end of the experiment.     

A comparison of AmBisome to amphotericin B for treatment of systemic candidiasis in very low birth weight infants

PURPOSE: Amphotericin B is considered the treatment of choice for systemic candidiasis, but adverse effects may limit its use. An alternative option for the treatment of candidiasis includes lipid preparations of amphotericin B. This study investigated the safety and efficacy of AmBisome, a lipid formulation of amphotericin B containing liposomal structures, for the treatment of systemic candidiasis in very low birth weight infants (VLBWI). MATERIALS AND METHODS: Data from 26 VLBWI treated with AmBisome in the study group (AmBisome group) from October 2003 to July 2006 were compared with data from 20 VLBWI treated with amphotericin B as a historical control (Amphotericin group). This study was a prospective, historical control, multi-center trial. RESULTS: Candida spp. was isolated in 73% (19/26) of the cases for the AmBisome group and 90% (18/20) of the cases for the Amphotericin group. The fungal eradication rate and the time to eradication was 84% (16/19) and 9+/-8 days in the AmBisome group, and 89% (16/18) and 10+/-9 days in the Amphotericin group, respectively (p=0.680 vs p=0.712). The major adverse effects were lower in the AmBisome group (renal toxicity, 21% vs 55%, p=0.029; hepatotoxity, 25% vs 65%, p=0.014, AmBisome group vs Amphotericin group, respectively). There was no significant difference in mortality attributed to systemic candidiasis (12% in the AmBisome group, 10% in the Amphotericin group, p=0.868). CONCLUSION: AmBisome is effective and safe for treating systemic fungal infections in VLBWI.     

Effects of modified amphotericin in experimental systemic candidiasis

Lysosomotropic composition of dialdehyde dextran and amphotericin B had a greater therapeutic effect in mice with systemic candidiasis compared to free amphotericin B. This composition normalized glucocorticoid function of the adrenal glands and decreased the severity of liver destruction at late terms of granulomatous inflammation. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 4, pp. 367–369, April, 2007     

Use of liposomal amphotericin B in the treatment of disseminated coccidioidomycosis

Disseminated fungal infection is an important cause of morbidity and mortality, especially in immunocompromised hosts. Amphotericin B is an important part of the therapy and treatment of invasive and life-threatening mycoses. We present a case of disseminated coccidioidomycosis in a patient who was successfully treated with liposomal amphotericin B (AmBisome) on steroid therapy. It appears that liposomal amphotericin B may offer an alternative and safe option in the treatment of coccidioidomycosis when conventional amphotericin B cannot be used.     

Amiloride prevents amphotericin B related hypokalaemia in neutropenic patients.

Twenty neutropenic patients with various haematological disorders were randomised prospectively to receive either intravenous amphotericin B alone or amphotericin B and oral amiloride 5 mg twice a day for treatment of confirmed or suspected fungal infection. Patients receiving amiloride had a significantly higher plasma potassium (p less than 0.01), a significantly lower urinary potassium loss (p less than 0.01), and required significantly less potassium chloride supplementation to maintain their plasma potassium within the normal range (p less than 0.001). Amiloride was well tolerated, had no clinically important side effects, and provided effective control of plasma potassium in patients treated with amphotericin B.     

Morphological changes in the brain of mice with systemic candidiasis treated with composition of amphotericin B and oxidized dextran

We observed morphological manifestation of encephalitis 3, 7, 10 and 28 days after intravenous infection of adult male CBA mice with Candida albicans. Compounds were administered intraperitoneally every other day starting from the next day postinfection. Untreated animals (100%) died over the period between days 18 and 20 postinfection; 60% animals receiving oxidized dextran alone survived by day 28 of observation. All animals treated with amphotericin B and composition of amphotericin B and oxidized dextran survived. On day 3 postinfection, the count of macrophage infiltrates and granulomas in the cerebral interstitium of mice treated with amphotericin B was equal to that in untreated mice, but was sufficiently lower in animals treated with the composition or oxidized dextran alone. On day 10, this index was similar in all groups and was approximately 5 times lower than in untreated animals on day 3. On day 28, macrophage infiltrates and granulomas were absent in the brain of all treated mice. These data suggest that oxidized dextran produced a therapeutic effect, which manifested earlier than the effect of amphotericin B and potentiated its effect, probably due to its competition with Candida albicans for mannose receptors on the brain-blood barrier endothelium.     

Determination of amphotericin B, liposomal amphotericin B, and amphotericin B colloidal dispersion in plasma by high-performance liquid chromatography.

Amphotericin B is a potent polyene antifungal drug for intravenous treatment of severe infections. It is used as amphotericin B-deoxycholate and in order to reduce amphotericin B toxicity as lipid-formulated complex (liposomal or colloidal dispersion). A sensitive and specific analytical method is presented for the separation of lipid-complexed and plasma protein-bound amphotericin B in human heparinized plasma. This separation, which is required for pharmacokinetic studies, is achieved by solid-phase extraction (SPE) via Bond Elut C18. The protein-bound amphotericin B has a higher affinity to the SPE material and is therefore retained, whereas the lipid-complexed amphotericin B is eluted in the first step. The recovery of the SPE was >75% for high concentrations and >95% for low concentrations. Quantification was performed by reversed-phase HPLC using a LiChrosorb-RP-8 column, UV detection (lambda=405 nm) and a mixture of acetonitrile-methanol-0.010 M NaH2PO4 buffer (41:10:49, v/v) as mobile phase. The retention time for amphotericin B under the given conditions was 6.7 min. The calibration curves were found to be linear (r > or = 0.999) in two different ranges (5.0-0.50 microg/ml and 0.50-0.005 microg/ml). Intra- and inter-day precision and accuracy fulfilled the international requirements. No interference from other drugs (typical broad medication for intensive-care patients) or common plasma components was detected in >400 samples analyzed.     

Differential effects of amphotericin B and liposomal amphotericin B on inflammatory cells in vitro

OBJECTIVE AND DESIGN: To understand whether the pseudo-allergic reactions to amphotericin B (AmB) administration are due to AmB or to the solubilizing vehicles, a study was designed to evaluate the effects of AmB, liposomal AmB, (L-AmB), AmB-deoxycholate micellar complex (AmB-DC) and deoxycholate (DC) on the responses of rat serosal mast cells (RSMC) and of human basophils (HB), in vitro. MATERIALS AND METHODS: Serosal mast cells were obtained by density gradient centrifugations from male Wistar albino rats. Partially purified HB were obtained from healthy donors. The cells were exposed to AmB, L-AmB, AmB-DC and DC. Histamine release was measured fluorometrically, and the release of lactic dehydrogenase (LDH) was measured spectrophotometrically. HB activation was evaluated cytofluorimetrically by CD63 expression. RESULTS: AmB and L-AmB did not evoke histamine or LDH release from either RSMC or HB. CD63 expression was not induced in HB challenged with AmB and L-AMB. On the other hand, AmB-DC and DC produced a cytotoxic histamine release from both RSMC and HB, and a sustained increase of CD63 expression on HB. CONCLUSIONS: Only AmB-DC was able to induce the release of inflammatory mediators from RSMC and HB. Conceivably, the cytotoxic effect is accounted for by the micellar complexes with DC, which has been confirmed as a powerful histamine releaser, and held responsible for the pseudo-allergic reactions after AmB-DC administration. The data lend support to a better safety profile of L-AmB.     

Successful treatment with liposomal amphotericin B in two patients with persisting fungemia

Two granulocytopenic patients in whom fungemia persisted despite therapy with deoxycholate amphotericin B were subsequently successfully treated by daily intravenous administration of amphotericin B entrapped in sonicated liposomes made of egg yolk lecithin, cholesterol and stearylamine in a molar ratio of 4:3:1. High serum concentrations of amphotericin B could be maintained in both patients during therapy with liposomal amphotericin B and were associated with high in vitro antifungal activity. Liposomal amphotericin B was tolerated much better than the deoxycholate preparation. These findings suggest that the liposomal amphotericin B preparation is superior in the treatment of fungemia in granulocytopenic patients, and that randomized trials are warranted.Research Associate, Fund for Medical Scientific Research, Belgium.     

Increased susceptibility to systemic candidiasis in interleukin-6 deficient mice.

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates multiple aspects of the innate immune response. It has been recently shown that endogenous IL-6 is crucial for an efficient defence against severe infections with Gram-negative and Gram-positive bacteria. The aim of the present study was to investigate the role of endogenous IL-6 in the defence against infection with the yeast Candida albicans. During experimental candidemia, IL-6 deficient mice (IL-6-/-) had a decreased survival and an increased fungal load in their organs when compared with IL-6+/+ controls, despite increased plasma concentrations of tumour necrosis factor-alpha (TNF), interleukin-1 alpha (IL-1 alpha) and IL-1 beta, IL-6-/- mice were not able to mount an efficient neutrophil response during the infection. When mice were rendered neutropenic by cyclophosphamide, neutropenic IL-6-/- mice were equally susceptible to C. albicans when compared to neutropenic IL-6+/+ mice, implying that neutrophils mediate the beneficial effect of endogenous IL-6. In conclusion, IL-6-/- mice are more susceptible to disseminated candidiasis, and the effect of IL-6 is most likely mediated by neutrophils.     

Prevention of nephrotoxicity of amphotericin B during the treatment of deep candidiasis

Previous observations suggest that tubulo-glomerular feedback could be involved in amphotericin B nephrotoxicity. We then investigated the influence of sodium status on the occurrence of renal damage during amphotericin B therapy. A retrospective survey demonstrated that impaired renal function occurred during therapy in 67 per cent of the patients who received amphotericin B alone and in 12 per cent of the patients who received amphotericin B and ticarcillin (parenteral sodium supplement of 100-150 meq per day). Prospective studies were then undertaken both in adults and children. Intravenous sodium supplement was given intravenously as routine prophylaxis with amphotericin B therapy. In all courses amphotericin B was successfully administered without deterioration in renal function. These results support the hypothesis that parenteral sodium supplementation reduces the frequency of developing impaired renal function during amphotericin B therapy.     

Use of liposomal amphotericin B in bone marrow transplant

Increasing number of transplants worldwide has resulted in an increase in the incidence of fungal infections. Prolonged neutropenia, immunosuppression and graft vs. host disease all result in high predisposition to fungal infections. The likelihood of developing a fungal infection increases with the severity and duration of neutropenia, which, in the case of cancer or chemotherapy for the treatment of hematological malignancies, can range from a few days to several weeks. Invasive fungal infections are difficult to diagnose and neutropenic patients with fever often receive empirical antifungal therapy. This provides a rationale for the prophylactic use of antifungal agents. The empirical use of liposomal amphotericin B has overcome some of the difficulties usually found in this setting. The majority of clinical efficacy data related to liposomal amphotericin B are derived from compassionate use studies and case series. The major advantage of these liposomal formulations of amphotericin B is a reduction in amphotercin toxicity. Use of liposomal amphotericin has been shown to be a cost-effective approach abroad and the same has been our experience also. Commercially ambisome and Fungisome are the only products that contain true liposomes. Unlike ambisome, which needs to be used in dose of 3 mg/kg/day Fungisome is effective in the dose of 1-3 mg/kg bodyweight. The Indian liposomal preparation has shown to be safe and effective used in over 150 transplant patients in our experience. We conclude that the liposomal amphotericin is better-tolerated and also gives,better responses in documented fungal infections.     

Treatment of deep mycoses with liposomal amphotericin B

Amphotericin B is the mainstay of therapy of many deep mycoses, but its use is seriously hampered by dose-limiting nephrotoxicity. In this study a liposomal formulation of amphotericin B was administered to ten patients with proven deep mycoses: invasive aspergillosis (n=4), deep candidiasis (n=4) and zygomycosis (n=2). The mean daily dosage of liposomal amphotericin B was 3.0 mg/kg (range 2.5 to 4 mg/kg), the mean total dosage of liposomal amphotericin B 2,781 mg (range 87 to 5,220 mg) and the mean duration of treatment 17 days (range 3 to 33 days). Treatment with liposomal amphotericin B was associated with little nephrotoxicity and an overall survival rate of 50 %. The median increase of serum creatinine from baseline levels was 0.38 mg/dl (–1.2 to 2.6 mg/dl).     

Suppression of immunological responses in mice by treatment with amphotericin B.

The polyene antibiotic amphotericin B (AmB) caused a marked suppression of the cell-mediated immune response in mice. Similar treatment did not effect the humoral antibody response. The immunosuppressive property of the drug was related to its ability to inhibit the manifestation rather than the induction phase of the delayed-type hypersensitivity response. In vitro AmB suppressed mitogen-induced lymphocyte proliferation. The drug seemed to act at the proliferative phase of the response. Results presented show that the T cell response was much more sensitive to the action of AmB than was the B cell response. During AmB chemotherapy consideration must be given to the immunosuppressive properties of this drug.     

Effect of liposomal amphotericin B on murine macrophages and lymphocytes.

The effect of liposome-encapsulated amphotericin B on mouse macrophages and on T- and B-lymphocyte functions in vitro was compared with that of free amphotericin B. Liposomal amphotericin B was generally less toxic than the free form of the drug. Low concentrations of free amphotericin B completely inhibited the serum-dependent induction of transglutaminase, a marker for macrophage differentiation, and production of superoxide anion by macrophages, whereas encapsulation of the drug within liposomes protected the cells from these adverse effects. Liposomal amphotericin B did not affect the blastogenic response of T cells compared with the free drug, which was inhibitory at high concentrations. Antibody production in vivo was inhibited partially by both free and liposomal amphotericin B. These results thus suggest that encapsulation of amphotericin B in liposomes reduces the immunosuppressive effects exerted by free amphotericin B. This provides further justification for therapeutic use of liposomal amphotericin B in systemic fungal infections (G. Lopez-Berestein, R. Mehta, R. L. Hopfer, K. Mills, L. Kasi, K. Mehta, V. Fainstein, M. Luna, E. M. Hersh, and R. L. Juliano, J. Infect. Dis. 147:939-945, 1983).     

Immunoprotection against systemic candidiasis in mice

We have previously described an immunosuppressive B cell mitogenic(ISM) protein, p43, produced by Candida albicans, which playsan important role in the survival of the microorganism in thehost. The N-terminal amino acid sequence of p43 was found tobe different from all amino acid sequences registered in updatedprotein databanks. Immunization of BALB/c mice with p43 partiallyneutralized the biological effects of this protein, namely depletionof bone marrow pre-B and B cells, the increased numbers of totaland large B and CD4⁺ lymphocytes, and the nonspecific polyclonalresponse of splenic IgG2a-, IgG2b- and IgM-secreting plaqueforming cells.immunization of BALB/c mice with p43 fully protectedthe mice against the fungal infection. In contrast, immunizationwith C. albicans sonicates (Cs) was not protective. Our dataindicated that specific antibodies against p43 protected, whereasthose against Cs facilitated C. albicans infection. Thus, theratio between anti-p43 and anti-Cs antibody titres was muchlower in the non-protected mice (Cs-immunized and control non-immunized)than in p43-immunized mice. Moreover, passive administrationof specific anti-p43 antibodies significantly protected againstfungal infection, whereas passive administration of specificanti-Cs antibodies markedly increased the susceptibility toC. albicans Infection. These observations are discussed on thebasis of alternative approaches of immunointervention.     

Mucosal and systemic candidiasis in congenitally immunodeficient mice.

Colony counts and light microscopy were used to assess the capacity of Candida albicans to colonize, infect the alimentary tract, and cause disseminated disease in athymic (nu/nu), euthymic (nu/+), beige (bg/bg), black (bg/+), beige athymic (bg/bg nu/nu), or beige euthymic (bg/bg nu/+) germfree mice. The alimentary tracts of all six genotypes of germfree mice were quickly colonized after exposure to yeast-phase C. albicans. Only bg/bg nu/nu mice showed obvious morbidity and mortality after mucosal colonization with C. albicans. Histopathology of C. albicans-colonized immunocompetent (nu/+, bg/+) and singly immunodeficient (nu/nu, bg/bg, bg/bg nu/+) mice showed minimal to moderate mucosal infections, whereas doubly immunodeficient (bg/bg nu/nu) mice showed extensive yeast and hyphal infection of the palate, tongue, esophagus, and stomach. A progressive systemic infection in C. albicans-colonized mice occurred only in bg/bg nu/nu mice 12 to 16 weeks after colonization and mucosal infection. Thus, it appears that a combination of defective cell-mediated immunity and phagocytic cell defects (polymorphonuclear leukocytes and/or macrophages) predisposed mice to severe mucosal and systemic candidiasis of endogenous origin. This is the first report of a mouse strain that is not only naturally susceptible to mucosal and systemic candidiasis of endogenous origin but also shows lethality at early (1 to 4 weeks) and late (12 to 16 weeks) times after alimentary tract colonization.     

Physiological and metabolic alterations accompanying systemic candidiasis in mice.

Mice challenged intravenously with 10(6) viable Candida albicans died between 1 and 16 days after infection. Near the time of death, over 98% of the recoverable fungi came from the kidneys. Physiologically, animals were in renal failure near the time of death as evidenced by elevated blood urea nitrogen (BUN) and blood creatinine levels and a creatinine clearance rate which was about one-half normal. No abnormalities in liver glucogen and blood glucose levels were detectable. When mice were challenged with 4.5 X 10(6) viable C. albicans, they all died within 12 h. Near the time of death they had normal BUN values and were hyperglycemic. In mice receiving 4.5 X 10(6) heat-killed C. albicans, no deaths occurred and liver glycogen, blood glucose, and BUN levels all remained within a normal range and were different from responses to bacterial endotoxin. Cumulatively, the results demonstrate two distinct syndromes for the pathogenesis of experimental C. albicans infections. At the lower dose, mice were in renal failure associated with progressive renal infection. At the higher dose, renal failure was not observed. If a toxin was associated with death from the latter dose, it was not similar to bacterial endotoxin.