George S. Kobayashi

Strategies in the Treatment of Systemic Fungal Infections

IN the past few years, there has been an increase in the number of systemic fungal infections seen in clinical practice. These infections have been of two types: those due to primary pathogens such...

Amphotericin B-resistant yeast infection in severely immunocompromised patients

Systemic yeast infections are a major cause of morbidity and mortality in severely immunocompromised patients. The in vitro susceptibility to amphotericin B of 29 yeasts causing fungemia was examined in 26 patients undergoing allogeneic or autologous bone marrow transplantation and/or myelosuppressive chemotherapy. The minimal inhibitory concentrations (MICs) of amphotericin B observed with blood isolates from these patients were significantly higher than those observed with blood, sputum, or skin isolates from non-immunocompromised patients (p less than 0.01). All episodes (10 of 10) of bloodstream infection in immunocompromised patients caused by isolates with MICs greater than 0.8 micrograms/ml were fatal, versus eight of 17 episodes of bloodstream infection caused by yeasts with MICs of 0.8 micrograms/ml or less (p = 0.04). Although 15 of 26 patients received empiric treatment with amphotericin B before laboratory evidence of fungemia developed, the amphotericin B susceptibilities of their isolates were not significantly different from those of patients who had not received empiric amphotericin B treatment. It is concluded that yeast fungemia in severely immunocompromised patients is often caused by organisms resistant to the usual concentrations of amphotericin B obtainable in vivo, and that this finding is clinically significant.

Classification of Polyene Antibiotics According to Chemical Structure and Biological Effects

Fourteen polyene antibiotics and six of their semisynthetic derivatives were compared for their effects on potassium (K+) leakage and lethality or hemolysis of either Saccharomyces cerevisiae or mouse erythrocytes. These polyene antibiotics fell into two groups. Group I antibiotics caused K+ leakage and cell death or hemolysis at the same concentrations of added polyene. In this group fungistatic and fungicidal levels were indistinguishable. Group I drugs included one triene (trienin); tetraenes (pimaricin and etruscomycin); pentaenes (filipin and chainin); one hexaene (dermostatin); and one polyene antibiotic with unknown chemical structure (lymphosarcin). Group II antibiotics caused considerable K+ leakage at low concentrations and cell death or hemolysis at high concentrations. The fungistatic levels were clearly separable from fungicidal. This group included the heptaenes (amphotericin B, candicidin, aureofungin A and B, hamycin A and B), and five of their semisynthetic derivatives (amphotericin B methyl ester, N-acetyl-amphotericin B, hamycin A and B methyl esters, and N-acetyl-candicidin). Nystatin, classified as a tetraene, and its derivative, N-acetyl nystatin, also were in this group.

Role of cell defense against oxidative damage in the resistance of Candida albicans to the killing effect of amphotericin B.

A laboratory-derived mutant of Candida albicans B311 (L) and a clinical isolate (C) of C. albicans, both lacking membrane ergosterol, were less susceptible to amphotericin B (AmB)-induced cell membrane permeability to K+ and lethality than was the wild-type laboratory strain (B311) which contained ergosterol. The resistance of L and C to AmB-induced killing was much greater than the level of resistance to AmB-induced cell membrane permeability. L and C were also less susceptible to killing by H2O2 than was B311, and when treated with menadione, they each produced less H2O2 than did B311. In addition, their levels of catalase activity were 3.8-fold (L) and 2-fold (C) higher than that of B311. The ergosterol deficiency in L and C probably impaired AmB binding to the cells, thereby lowering AmB effectiveness as measured by both cell membrane permeability and killing. Resistance of strains L and C to oxidation-dependent damage likely contributed to a diminished response to AmB-induced lethality.

Therapy of Murine Aspergillosis with Amphotericin B in Combination with Rifampin or 5-Fluorocytosine

Suboptimal doses of amphotericin B in combination with either rifampin or 5-fluorocytosine were better than single-drug therapy in the treatment of disseminated Aspergillus fumigatus infection in mice. Despite the increased effectiveness of combination therapy, none of the therapeutic regimens we used completely eradicated infections in the mice when evaluated by mycological culture, even in long-term survivors.

Activity of Amphotericin B, 5-Fluorocytosine, and Rifampin Against Six Clinical Isolates of Aspergillus

Amphotericin B in combination with 5-fluorocytosine was synergistic against three clinical isolates of Aspergillus fumigatus and one of three clinical isolates of A. flavus. Amphotericin B in combination with rifampin was synergistic against all six clinical isolates of Aspergillus tested. The levels of 5-fluorocytosine and rifampin required for synergism were higher than clinically achievable concentrations when measurements of synergism were based on visual turbidity; but when the effects of the drugs were measured by inhibition of ribonucleic acid synthesis or dry-weight increase, much lower concentrations were effective.

Molecular Basis for the Selective Toxicity of Amphotericin B for Yeast and Filipin for Animal Cells

Among the polyene antibiotics, many, like filipin, cannot be used clinically because they are toxic; amphotericin B, however, is useful in therapy of human fungal infections because it is less toxic. Both the toxicity of filipin and the therapeutic value of amphotericin B can be rationalized at the cellular and molecular level by the following observations: (i) these polyene antibiotics showed differential effects on cells; filipin was more potent in lysing human red blood cells, whereas amphotericin B was more potent in inhibiting yeast cell growth; and (ii) the effects of filipin were more efficiently inhibited by added cholesterol, the major membrane sterol in human cells, whereas the effects of amphotericin B were more efficiently inhibited by ergosterol, the major membrane sterol in yeast. The simplest inference is that the toxicity and effectiveness of polyenes are determined by their relative avidities for the predominant sterol in cell membranes.

Hsp70 in parasites: as an inducible protective protein and as an antigen

The heat shock (HS) response is a general homeostatic mechanism that protects cells and the entire organism from the deleterious effects of environmental stresses. It has been demonstrated that heat shock proteins (HSP) play major roles in many cellular processes, and have a unique role in several areas of cell biology, from chronic degenerative diseases to immunology, from cancer research to interaction between host and parasites. This review deals with thehsp70 gene family and with its protein product, hsp70, as an antigen when pathogens infect humans. Members of HSP have been shown to be major antigens of many pathogenic organisms when they experience a major temperature shift upwards at the onset of infection and become targets for host B and T cells.

A temperature-sensitive strain ofHistoplasma capsulatum has an altered Δ9-fatty acid desaturase geneacid desaturase gene

We have isolated and characterized the Δ9-desaturase gene (Ole1), which codes for a key enzyme involved in regulating membrane fluidity in animal cells and microorganisms, from two strains ofHistoplasma capsulatum, one that is temperature-tolerant (G217B) and the other temperature-susceptible (downs). These pathogenic fungi are dimorphic in that they undergo a morphologic transition from the mycelial to yeast-like form when the temperature of incubation is switched from 25 to 37°C or when they infect a susceptible host. The coding sequences of the two genes, both containing an intron of 93 nucleotides, arevirtually identical and analogous to the Δ9-desaturase gene ofSaccharomyces cerevisiae and those of the rat, mouse and human.Ole1 transcription of the thermotolerant G217B and thermosensitive Downs strains is similar in yeast phase cells and during the temperature shift down from 34, 37, or 40 to 25°C (yeast-to-mycelia transition). Nevertheless, Δ9-desaturase gene is transcriptionally inactive in mycelia of G217B at 25°C while it is actively transcribed in the Downs strain at the same temperature. These results are in agreement with the finding that membranes of the Downs strain have a higher level of oleic acid. The differential expression of Δ9-desaturase genes is discussed in relationship to differences in thermosensitivity in the fungal isolates and in regulating the level of expression of heat shock genes.

Demonstration of antigenemia in patients with invasive aspergillosis by solid phase (protein A-rich staphylococcus aureus) radioimmunoassay

A solid phase radioimmunoassay (RIA) was used to detect Aspergillus antigenemia in three patients, two with autopsy proved disseminated aspergillosis and one with a suspected infection. These RIA studies suggest that screening for antigenemia may be a specific and sensitive diagnostic test for disseminated aspergillosis.