Nystatin-intralipid preparation: characterization and in vitro activity against yeasts and molds.

The objective of our studies is the development of a novel formulation of nystatin (NYT) that could be administered systemically and might be used for therapy of invasive mycoses. We developed a formulation of nystatin and intralipid (IL), which is a clinically used food supplement, and this report focuses on the characterization of NYT-IL, assessment of its antifungal activity and in vitro toxicity. We characterized physical properties of the NYT-IL preparation and its stability during storage. Susceptibility of Candida, Aspergillus and Fusarium species was determined using a CLSI technique. In vitro toxicity of NYT-IL was assessed using an assay measuring hemolysis of sheep red blood cells (SRBC) and leakage of potassium. It was found that: (1) the particle size in NYT-IL did not differ from that of IL; (2) over 80% of NYT was in association with IL; and (3) these features did not change during storage. All Candida and Aspergillus strains had lower minimal inhibitory concentration (MIC) values for NYT-IL than that for NYT; the MICs of the Fusarium strains were similar for NYT & NYT-IL. Toxicity assays showed that the NYT-IL formulation is less toxic than NYT. In conclusion, we describe a novel, characterized, stable formulation of nystatin, nystatin-intralipid, with in vitro activity against pathogenic Candida and Aspergillus species.

Dementia and myoclonus in a case of cryptococcal encephalitis.

Dementia and generalized myoclonic jerks were the only neurologic features in a patient with cryptococcal encephalitis. Despite the presence of numerous budding yeasts identified as Cryptococcus neoformans in the CSF, there was no inflammatory reaction. Protein and glucose levels were normal, with no pleocytosis.

The identification and tracking of Candida albicans isolates from oral lesions in HIV-seropositive individuals.

Restriction fragment polymorphism analysis was used to investigate the identity and genotypic relatedness of Candida albicans strains isolated from human immunodeficiency virus (HIV)-infected patients with or without oral candidiasis and from some of their sexual partners. Use of the species-specific DNA probe Ca3 revealed that most subjects carried a single distinct C. albicans strain throughout the course of the study, during both symptomatic and asymptomatic periods. Sexual partners were more likely to carry the same or similar C. albicans isolates than unrelated subjects, raising the possibility of transmission via intimate contact. One patient appeared to acquire his partner's isolate, which then became predominant in both partners in subsequent isolations. These findings indicate that recurrent oral candidiasis is usually caused by a single persistent strain unique to each patient, but that in some cases transmission via intimate contact may occur between sexual partners.

Synthesis and characterization of novel water soluble amphotericin B-arabinogalactan conjugates.

The coupling of amphotericin B (AmB), a water-insoluble antifungal agent, to arabinogalactan (AG) via an imine or amine bond was systematically investigated. AG was oxidized using potassium periodate, purified from the oxidizing agent using ion-exchange chromatography, and reacted with AmB to form the Schiff base. The Schiff base was reduced to the amine using borohydride. All reactions took place in aqueous media. The purification of the oxidized AG from the oxidizing agent was essential to prevent oxidative degradation of AmB at the coupling step. We investigated the effects of AmB to AG ratio, buffer type, and reaction pH on the reaction yield, molecular weight, conjugate activity against pathogenic yeast and hemolytic activity. The optimum coupling conditions were buffer borate 0.1 M, pH 11 at room temperature for 48 h. Lower toxicity in vivo was achieved by using low-pressure gel permeation chromatography and applying the solution of AmB-AG conjugate through a Sephadex column. Both amine and imine AmB-AG conjugates were soluble in water and exhibited improved stability in aqueous solutions as compared to the unbound drug. The conjugates showed comparable minimum inhibitory concentration (MIC) values against Candida albicans. The conjugates were about 60 times less hemolytic against sheep erythrocytes than the free drug, and about 40 times less toxic in BALB/c mice.

Cytokine-induced resistance to microbial infections in normal, immunosuppressed and bone marrow transplanted mice.

We studied the efficacy of in vivo and in vitro treatments with IL-1, IL-2, IL-3, and GM-CSF in the protection against bacterial (Salmonella typhimurium), fungal (Candida albicans) and viral (influenza virus A/PR8) infections, of normal, sublethally irradiated and lethally irradiated, bone marrow (BM) reconstituted mice. In parallel, the cytokines were tested for their ability to potentiate hematopoietic activity in vitro and in vivo. We demonstrate that, under the experimental conditions employed, IL-1 had the best protective activity against the three micro-organisms in both normal and immunocompromised mice when administered in vivo. Administration of IL-2 led to increased resistance in normal but not in immunodeficient mice, whereas GM-CSF had no beneficial effects. In contrast, preincubation of BM cells in these cytokines, singly or combined, prior to transplantation to lethally irradiated mice, did not confer protection against subsequent infection, although it increased the number of BM derived CFU-GM in culture (except in the case of IL-2). Administration of IL-1 or GM-CSF to BM transplanted mice facilitated WBC recovery, whereas IL-2 delayed it. Collectively, the data suggest that IL-1, alone or combined with other cytokines, may be beneficial in the prevention or treatment of microbial infections in immunocompromised and BM transplanted patients. It can also be concluded that enhanced hematopoietic recovery may not always coincide with the development of resistance to micro-organisms.

Disseminated visceral fusariosis treated with amphotericin B-phospholipid complex.

Fusariosis, a rare infectious disease of the immunocompromised host, is relatively resistant to amphotericin B (AmB) or other antifungal agents. We describe a 5-year follow-up of a 40 year old woman with T-type acute lymphoblastic leukemia who following chemotherapy developed prolonged high fever, chills, night sweats, and severe weakness. Liver function tests were impaired and abdominal computerized tomography (CT) showed multiple lesions in the liver and abnormal structure of the spleen. A laparotomy revealed multiple granulomas containing Fusarium sp. in the liver, and the spleen was heavily infiltrated by the same fungus. The patient failed to respond to the conventional AmB dosage form (Fungizone) even after a total dose of 3.0 g was given, and developed significant renal impairment. AmB was complexed (in a mole ratio of 1:16) with a mixture of the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol (mixed in 7:3 mole ratio). The resulting drug complex, AmB-PLC, was then administered (1-4 mg/kg/day, total dose 4.2 g) and subsequently the patient was cured of all symptoms of fusariosis. There were only mild side effects and no nephrotoxicity was evident. On the contrary, marked improvement of the renal function tests occurred during AmB-PLC treatment. Eight months later, she developed a spinal lesion with dense consistency in L5 and S1, and after receiving another course of AmB-PLC (3.1 g) she recovered completely. In a 2 year follow-up period the patient had no further relapse of the fungal disease. Subsequent chemotherapy given for relapse of the leukemia was followed by a new fungal infection, which was treated with AmB-cholesteryl sulfate complex (Amphocil).(ABSTRACT TRUNCATED AT 250 WORDS)

Increasing fungal isolation from clinical specimens: experience in a university hospital over a decade.

The local patterns of fungal isolates were studied by a retrospective analysis of fungal species isolated from clinical specimens in a university hospital in Jerusalem. Between 1984 and 1993, 5630 fungi [4071 patient unique isolates (PUI)] were isolated and identified. During the study decade, the annual incidence of all isolates increased 2.7-fold, and PUI increased 1.6-fold. Candida albicans accounted for 61% of PUI; urine was the source of 53%. The intensive care units (ICUs) and the Bone Marrow Transplantation (BMT) Department had the highest incidence of fungal isolation. The following trends were observed: (1) a decrease in the relative frequency of C. albicans and increase in Candida tropicalis; (2) increased number of isolates from urine, surgical wounds and intra-abdominal sites; (3) increased number of isolates from ICUs and BMT. Fungi are emerging as important hospital-acquired pathogens in tertiary care and teaching hospitals, and are associated with high rates of morbidity and mortality. It is important to be familiar with the local patterns of fungal isolation in order to improve treatment.

Invasive mucormycosis in a non-immunocompromised patient.

Primary invasive cutaneous mucormycosis in a non-immunocompromised patient is described. Cultures from the infected tissues yielded a zygomycete which was identified as Saksenaea vasiformis. The patient was successfully treated with debridement of the lesion and amphotericin B.

Altered gut barrier function to Candida during parenteral nutrition.

We hypothesize that catheter-related sepsis with Candida during total parenteral nutrition (TPN) is caused by Candida translocation from the gut. Fifty male Sabra rats weighing 330 +/- 40 g were randomized into four groups and put into metabolic cages: group 1 (n = 16), nonoperated free-feeding controls; group 2 (n = 10), infused with normal saline and free feeding; group 3 (n = 14), infused with TPN solution for a total of 36 kcal and 1.5 g g protein.100 g-1 body wt.day-1;group 4 (n = 10), same TPN regimen as group 3 but also receiving oral and intravenous antibiotics. On day 7, all animals received 1.5 x 10(10) viable Candida albicans CBS 562 cells by gavage, and 24 h later, the number of Candida colony-forming units in blood, mesenteric lymph nodes, and kidneys was determined. No growth of Candida was detected in group 1 or group 2. Positive Candida cultures were found in the blood, mesenteric lymph nodes, and kidneys of groups 3 and 4, although levels reached statistical significance only for mesenteric lymph nodes in group 3. Because Candida growth occurred exclusively in groups receiving TPN and bowel rest, we conclude that altered gut-barrier function to Candida occurs during TPN and speculate that Candida sepsis during TPN might be the result of Candida translocation from the gut due to the combination of high-density Candida colonization and favorable local conditions in the gut induced by TPN and bowel rest.

Synthesis and antifungal activity of medicagenic acid saponins on plant pathogens: modification of the saccharide moiety and the 23 alpha substitution.

The study of structure-antifungal activity relationships of medicagenic acid saponins was widened to include synthetic glycosides of mannose, galactose, cellobiose, and lactose as well as a 23 alpha-hydroxymethyl analog of medicagenic acid, namely, methyl 2 beta,3 beta-dihydroxy-23 alpha-hydroxymethyl-delta (12)-oleanene-28 beta-carboxylate, against Sclerotium rolfsii, Rhizoctonia solani, Trichoderma viride, Aspergillus niger, and Fusarium oxysporum. The native glucose-containing saponin was a more effective antifungal agent than the aforementioned saponins, except in the case of the cellobiose-containing derivative and F. oxysporum. A carboxyl substituent at the 23 alpha position of the sapogenin brought about higher fungistatic activity than a methyl carboxylate which, in turn, was more effective than an hydroxymethyl group at the same position.

Conjunctival oculosporidiosis in east Africa caused by Rhinosporidium seeberi.

BACKGROUND AND OBJECTIVE: Conjunctival rhinosporidiosis is an infectious disease rarely recorded outside the Indian subcontinent. The disease is caused by Rhinosporidium seeberi, an endosporulating microorganism of uncertain taxonomic classification. We report a series of cases manifesting this infection. MATERIAL AND METHODS: The demographic, clinical, and histopathologic data of 14 cases of conjunctival rhinosporidiosis on record at our Ophthalmic Pathology Laboratory were reviewed. RESULTS: All cases were from East Africa; 10 were from Malawi and 4 from Kenya. Ten specimens were from males and three from females. Their age ranged from 7 to 20 years. All patients were treated by surgical excision, and no recurrence was recorded. None of the cases was diagnosed clinically as rhinosporidiosis. Histologically, all stages of the organism's life cycle could be found in the excised tissue, from small trophocytes to large sporangia-containing sporoblasts. There were changes in histochemical stainings with growth and maturation. In general, the inflammation was of chronic nongranulomatous type. CONCLUSIONS: Conjunctival rhinosporidiosis is a rare infectious disease that typically appears in young males in rural regions, and that can be treated by surgical excision. It typically causes chronic nongranulomatous inflammation. Various stages of the R seeberi life cycle can be seen in the affected tissue.

Saponins as antimycotic agents: glycosides of medicagenic acid.

The continuous search for new antimycotic drugs is a consequence of the broad use of immunosuppressive drugs and broad-spectrum antibiotics, high number of AIDS patients, and widespread dermatophyte infections. The concern with increased resistance due to widespread and prolonged antifungal treatment, particularly with azoles, is noteworthy. Our efforts were focused on medicagenic acid derivatives isolated from alfalfa and on semisynthetic ones. In general, these materials exhibited potent fungistatic effects against several plant pathogens and human dermatophytes. Furthermore, they were fungicidal against medically important yeasts, showing a most impressive activity against Cryptococcus neoformans, the minimal fungicidal concentration (MFC) value of the gluco derivative of medicagenic acid, compound G2, is 4 micrograms/ml. The mode of action as well as the structure-activity relationships of these compounds were studied. Compound G2, when applied topically, was effective in curing skin lesions of guinea pigs infected with the dermatophyte Trichophyton mentagrophytes and good skin tolerance to the drug was noted. Furthermore, it had a life-prolonging effect on mice infected with C. neoformans and recently, liposomes containing compound G2 were used efficiently as a drug delivery system in treatment of murine cryptococcosis and candidiosis.

Activity of an Intralipid formulation of nystatin in murine systemic candidiasis.

Since nystatin (NYT) is used only topically owing to its toxicity upon systemic administration, a study was initiated aiming to develop a formulation of NYT that could be used systemically against invasive mycoses. The present research is a continuation of previous in vitro investigation of the antifungal effect of nystatin-Intralipid (NYT-IL) against Candida, exploring its in vivo activity. NYT-IL was tested in murine systemic candidiasis induced in naïve as well as cyclophosphamide-immunosuppressed female ICR mice. The infection was assessed by survival rate (SR), mean survival time (MST) and qualitative and quantitative fungal organ colonisation. Mice were treated by intravenous administration of various doses of NYT-IL for 5 consecutive days starting either 24h or 48 h after the initiation of infection. The experiments showed that NYT-IL is therapeutically effective in the murine candidiasis model. NYT-IL was found to be less toxic in vivo than NYT and therefore higher doses of NYT-IL could be used. The efficacy of NYT-IL was expressed in treated naïve and immunosuppressed mice by increased SR, prolonged MST and reduced fungal organ colonisation. Early initiation of treatment increased efficacy. In summary, the Intralipid formulation of NYT can be administered parenterally and is effective against systemic experimental Candida infection.

High power accelerator-based boron neutron capture with a liquid lithium target and new applications to treatment of infectious diseases.

A new conceptual design for an accelerator-based boron neutron capture therapy (ABNCT) facility based on the high-current low-energy proton beam driven by the linear accelerator at SARAF (Soreq Applied Research Accelerator Facility) incident on a windowless forced-flow liquid-lithium target, is described. The liquid-lithium target, currently in construction at Soreq NRC, will produce a neutron field suitable for the BNCT treatment of deep-seated tumor tissues, through the reaction (7)Li(p,n)(7)Be. The liquid-lithium target is designed to overcome the major problem of solid lithium targets, namely to sustain and dissipate the power deposited by the high-intensity proton beam. Together with diseases conventionally targeted by BNCT, we propose to study the application of our setup to a novel approach in treatment of diseases associated with bacterial infections and biofilms, e.g. inflammations on implants and prosthetic devices, cystic fibrosis, infectious kidney stones. Feasibility experiments evaluating the boron neutron capture effectiveness on bacteria annihilation are taking place at the Soreq nuclear reactor.

Pichia farinosa bloodstream infection in a lymphoma patient.

We describe a case of Pichia farinosa bloodstream infection in a lymphoma patient. Phenotypic methods failed to identify the isolate, which was identified by sequence-based methods. This case highlights the importance of implementing molecular methods for the identification of rare fungal pathogens.

Creatinine metabolism in Cryptococcus neoformans and Cryptococcus bacillisporus.

The pathogenic species of Cryptococcus, C. neoformans and C. bacillisporus, utilized creatinine as a source of nitrogen but not of carbon. Chromatographic and autoradiographic studies suggest that creatinine metabolism in both species involves a single step resulting in the production of methylhydantoin and ammonia. The enzyme responsible for this step, creatinine deiminase, was produced by the cells only in the presence of creatinine in both species. The synthesis of creatinine deiminase was repressed by ammonia in C. neoformans, but not in C. bacillisporus. A possible explanation for this variation, based on the ecological differences between the two species, is discussed. A novel method for measuring creatinine deiminase activity is also described.