POSTER SESSIONS Data are available for 14 patients and treated for more than 16 weeks. These 14 patients ageing from 20 to 49 years (mean 29.1 years, SD: 8.8) have a duration of disease prior to therapy between 3 and 54 months (mean 22.3 months, SD: 18.7). Twelve patients were de novo and two were recurrences. A mycological confirmation of Madurella mycetomatis infection was achieved in nine patients; three patients were positive for Leptosphaeria senegalesis, and in two patients no fungal identification was possible. At baseline all patients had tumefaction, 13 patients emission of grains, and 13 patients open sinus. Nine patients have completed the treatment: four were cured (absence of signs and symptoms and negative mycological exam after 40–48 weeks of treatment), four markedly improved after 48 weeks of treatment, and in one patient the disease progressed. One patient is still under treatment (24 weeks) with a clear improvement of the infection with disappearance of tumefaction and emission of grains. Four patients discontinued: one after 16 weeks of treatment due to being lost to follow-up, two due to laboratory abnormalities (after 16 and 40 weeks, respectively), and one because of exacerbation of the disease.

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MOPS at pH 7. Inocula were performed by spectrophotometric procedure and the turbidities of conidial suspensions were measured at 530 nm to 75-82 percentages of transmittance range and also haemotocymeter conidia recuent were done to obtain a concentration of 0,4-5,0 x 106 CFU/ml. The microtitration plates were incubated at 35°C and the MIC was read at 72 and 96 h. The MIC for AF was defined as the lowest concentration of the drug that inhibited all visible growth and for the antifungals the MIC was the lowest concentration of the drug that inhibited 50% of the growth (MIC 2) with respect the growth control. Results: we investigated the morphology in detail, and found that part of these species can be distinguished by conidiophore characters. The alignment of the rDNA regions and the tree based on these data showed dissimilarity of these strains originally identified as Exophiala spinifera. The MIC were: (0,25-4) for AB, (8-64) for FCZ, (0,063-0,5) for TF, (0,0311,25) for ITZ and (0,063-0,5) for VCZ. Conclusion: The antifungal drugs tested were active for all of these strains, exept for FCZ which is inactive against most filamentous fungi. These results suggests that these strains are new potential pathogens species because they are closely related to the pathogenic species Exophiala spinifera.

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POSTERS __________________

Taxonomy P1-002. Identification of clinical Fusarium isolates by PCR and direct sequencing Gutai A, Tintelnot K, Lemmer K. Robert Koch Institut, 13353 Berlin, Germany Isolates of the genus Fusarium can hardly be discriminated on a species level by marcro- and micromorphology and physiological parameters, even when they belong to related genera, f.e. Cylindrocarpon or Neocosmospora. Therefore 13 clinical isolates from bloodculture, mycetoma, skin, cornea and tracheal secretions have been studied by molecular methods. Five of the isolates previously had been identified as F. oxysporum, 6 as Fusarium sp. and two as Cylindocarpon lichenicola. All of them have been re-identified by PCR and sequencing of the internal transcribed spacer (ITS) region. Fungal DNA was extractedby sonification and amplified by ITS 4 and ITS 5 primers described by White et al. (1). The obtained sequences were aligned to NCBI GenBank sequences using the BlastN 2.0 program. Results: Four of the five F. oxysporum isolates were confirmed by sequencing, one was reidentified as F. proliferatum. Two of the six Fusarium sp. were also identified as F. oxysporum, two as Nectria haematococca (teleomorph of F. solani), one as F. solani and one as F. profileratum. The two Cylindrocarpon lichenicola isolates turned out to be F. solani. These results indicate that sequencing of isolates belonging to the family Hypocreaceae (Fusarium, Cylindrocarpon, Acremonium) leads to an identification superior to identification based on micromorphology alone. The ITS region of the rDNA seems to be discriminating enough between the two clinical most important Fusarium species, F. oxysporum and F. solani, but F. solani cannot be seperated enough from allied species, f.e. Necosmospora vasinfecta. Studies will be continued. (1) White T.J., Bruns T., Lee S. et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis N, Gelfand J., White T, PCR Protocols: A Guide to Methods and Applications; Academic Press, New York 1990; 315-322.

P1-003. New, potential pathogenic species of Exophiala spinifera complex Vitale R1,2, Verweij P2, De Hoog S1. 1Centraal Bureau voor Schimmelcultures, Baarn and 2University Medical Center Nijmegen, Nijmegen, The Netherlands Exophiala spinifera is a black yeast with very characteristic morphology: large, spine-like conidiophores. In a recent sequencing paper (Stud. Mycol. 43, 1999), we noticed that a few strains morphologically identified as Exophiala spinifera have very different ITS sequences. The aim of this present study was to introduce these fungi as new taxa and to develop phenetic markers for their identification. For this purpose the following steps were performed: DNA extraction, PCR, DNA purification, ITS sequencing using ITS1 and ITS4 as primers and tree construction based on confidently aligned regions. Data on nutrional physiology and tolerance tests are underway. The in vitro susceptibility tests by microdilution broth method according to NCCLS against amphotericin B (AB), fluconazole (FCZ), itraconazole (ITZ), terbinafine (TF) and voriconazole (VCZ) were also performed in RPMI media buffered with

P1-004. Dimorphic fungi, antifungal agents and phylogenetics Michael R. McGinnis. Department of Pathology, University of Texas Medical Branch, Galveston, Texas The thermally regulated dimorphic fungi Blastomyces dermatidis (Onygenaceae), Coccidioides immitis (Onygenaceae), Histoplasma capsulatum (Onygenaceae), Paracoccidioides brasiliensis (Onygenaceae), Penicillium marneffei (Trichocomacceae) and Sporothrix schenckii (Ophiostomataceae) were compared to Aspergillus fumigatus (Trichocomaceae), Pseudallescheria boydii (Microascaceae) and Scedosporium prolificans (Microascaceae). The dimorphic fungi were homogenous in their sensivities to the ranges (in brackets) tested for voriconazole (0,03-8 µg/ml), fluconazole (0,03-64 µg/ml), ketoconazole (0,03-8 µg/ml), miconazole (0,03-8 µg/ml), terbinafine (0,03-8 µg/ml), amphotericin B (0,03-2µg/ml), benomyl (2 µg/ml) and cyclohximide (0,4 µg/ml). A. fumigatus, which was derived from the penicillia, exhibits extremely high geometric mean MIC values for fluconazole (more than 64 µg/ml), terbinafine (1.68 µg/ml) and ketoconazoel (more than 8 µg/ml) that exeed the dimorphic fungi. S. schenkii is the only dimorphic fungus that had a similar geometric mean MIC to A. fumigatus for fluconazole (59.4 µg/ml). P. boydii geometric mean MICs for voriconazole (0.26 µg/ml) and itraconazole (0.89 µg/ml) were similar to those for miconazole (0.12 µg/ml) and ketoconazole (0.75 µg/ml), the latter two drugs beeing most often used to treat infections caused by this fungus. S. prolificans exhibited the highest mean MIC values to the drugs tested. In addition, P. boydii and S. prolificans, in contrast to the other fungi, were sensitive to cycloheximide and resistant to benomyl. Antifungal activity can be correlated to the phylogenetic relationships of the fungi studied. __________________

Antifungal Drugs and Therapy P2-001. Antifungal activity of P. aeruginosa and diphteria toxin Meltem Y Cirak, Semra Kustimur, Ayse Kalkanci, Halil Mansuroglu. Gazi University Faculty of Medicine Microbiology Department, Ankara, Turkey Studies concerning the antifungal activity of Pseudomonas aeruginosa, which was first assessed in 1994, have been steadily increasing. The antifungal activity of P. aeruginosa strains and Corynebacterium diphteria toxin on various Candida species were determined. The mechanism of the antifungal product of P. aeruginosa is still unknown. On the case that the mode of action of this antifungal product my have been inhibiting the elongation factor-2 (EF-2) and the protein synthesis like mechanism of exotoxin of the P. aeruginosa; the C. diphteria toxin which has the same activity mechanism has also been investigated for the antifungal activity. In vitro susceptibility studies revealed significant inhibition by 9 strains of P. aeruginosa, a standard strain P. aeruginosa ATCC 27853 and C.diphteria toxin on 6 strains of fungi known to infect humans; these were Candida albicans, Candida tropicalis, Candida parapsilosis, Candida kefyr, Candida rugosa, Candida guilliermondii. There had been no antifungal activity determined on these Candida strains in this study with diphteria toxin. The antifungal activity of P. aeruginosa strains were found to be 60% complete inhibition on C. albicans, 10% partial and 50% complete inhibition on C. parapsilosis, 10% complete and 50% partial inhibition on C. rugosa, 30% complete and 50% partial inhibition on C. guilliermondii, 30% complete and 40% partial inhibition on C. tropicalis, 50%complete and 10% partial inhibition on C. kefyr species.

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P2-002. A new regimen of antifungal prophylaxis in neutropenia S Antonopoulou, H Rodriguez, M Paesmans, M Husson, F Crokaert, M Aoun. Department of Microbiology and Infectious Diseases, Institute Jules Bordet. Brussels, Belgium. Objective: To compare the efficacy, tolerance and toxity of fluconazole associated to aerosolized amphotericin B (FLU+AAMB) with the oral suspension of itraconazole (ITR), as antifungal prophylaxis in neutorpenic patients with hematological malignancy (HM) or bone marrow transplantation (BMT). Method: It is a prospective randomized trial. Patients were eligible if they had HM or BMT with neutropenia. Exclusion criteria were known allergy to imidazoles or to amphotericin B, history of astma, respiratory failure and active alveolar hemorrhage. Surveillance fungal cultures were obtained before enrollment and subsequently 2/week including oral nose, skin and rectal swabs, urine, sputum and stool samples. Candida spp. were identified by Chromagar technique and API ID32C. Results: 83 successive patients were randomized (39 in FLU+AAMB arm and 44 in ITR arm) with similar characteristics in sex, age and predispossing disease. There were 23 BMT in FLU+AAMB arm and 27 in ITR. The mean time of neutropenia was 11 (3-52) days in FLU+AAMB and 15 (5-51) in ITR group. Overall 52 episodes of fever occured during prophylaxis. 3 patients developed superficial fungal infection (2 proven and 1 possible) and 7 patients developed invasive fungal infectioins (1 candidemia by C. parapsilosis and 2 pulmonary aspergillosis, 1 probable and 3 possible aspergillosis). Amphotericin B treatment was initiated in 9 cases (11%). 4 patients died (5%). Tolerance was similar between the two groups but toxity was higher in ITR (5% vs 0%). Les compliance was obtained with ITR (52% discontinuations on ITR vs 44% on FLU+AAMB). Conclusions: These preliminary results suggest that efficacy of prophylaxis is acceptable with both regiments altough more cases are necessary to determine the best choice. The compliance is poor in both arms and ITR is more toxic and less well tolerated. Antifungal prophylaxis reduced the fungal colonizatioin with Candida albicans during hospitalisation, but produced a shift through non albicans Candida spp. P2-003. Concentration of voriconazole in cerebrospinal fluid (CSF) in patients with cerebral mould infections Lutsar I, Troke P, Clark M. Pfizer Ltd., Central Research The prognosis of patients with cerebral mould infections is poor and mortality often exceeds 75%. Voriconazole is a new triazole antifungal agent that is active against a wide range of yeast and mould fungi, including Aspergillus spp, and Scedosporium spp. Compared with amphotericin B its lipophilicity is higher and this may result its greater penetration into CSF. The aim of the present study was to measure voriconazole concentrations in CSF in patients with cerebral fungal infections. Methods: Intravenous voriconazole was administered for the treatment of cerebral aspergillosis or scedosporiosis in two loading doses of 6 mg/kg/q12h. followed by a maintenance dose of 4 mg/kg/q12h. Steady state plasma voriconazole concentrations were achieved in 24 hours of therapy. CSF samples were collected at steady state from patients undergoinglumbar or ventricular punctures for therapeutic or diagnostic purposes. CSF samples were centrifuged, supermatant was stored at -20ºC, and analysed at specified labatory. Concentrations of voriconazole were determined using a fully validated high-performance liquid chromatography method with UV detection. Samples collected between 1 and 3 hours after end of infusion were considered to reflect peak concentrations. Penetration of voriconazoel into CSF was calculated as the ratio of mean CSF and plasma concentrations. Results: A total of 34 CSF samples were collected at different time post dose from 9 patients (6 aspergillosis and 3 scedosporiosis) aged from 2 to 50 years, Voriconazole concentrations in CSF ranged between 160 and 3925 ng/ml (median 755 ng/ml). Median CSF peak and through voriconazole concentrations were 2413 ng/ml and 789 ng/ml, respectively. The ratio of mean voriconazole CSF to plasma concentrations was 0.2. Conclusion: Voriconazole penetrates the CSF well yielding concentrations that exceed the MICs of most Aspergillus and Scedosporium species. Whether this will improve outcome of cerebral mould infections warrants further investigation. P2-004. First administration into man or UR-9825: A new triazole class of antifungal agent I Izquierdo, C Lurigados, I Perez, E Turmo, J Ramis. Research Area, J Uriach & Cía, Barcelona, Spain Ur-9825 is a new triazole class, with a potent broad espectrum antifungal activity. It is expected to be effective in treating patients suffering from all of the key opportunistic fungal infections including aspergillosis. A first-time-in-man, phase I, double-blind, randomised, placebo controlled, single rising dose study was conducted to evaluate the safety and pharmacokinetic profile of unchanged UR-9825. Seventy-two healthy volunteers were consecutively enrolled in-groups of 8 and randomly

assigned to receive UR-9825 or placebo. The following dose levels were studied: 5, 10, 20, 40, 80, 160, 240, 320 and 400 mg. All subjects gave their informed consent, and the study was approved by the Regional Ethical Committee. Ur-9825 was very well tolerated at all dose levels an no serious adverse effects were reported. Furthermore, no clinical significant trends in safety parameters were noted in vital signs, ECGS and labatory tests, UR-9825 is rapidly absorbed reaching Cmax values at 2-4 h and widely distributed throughout bodyfluids and the apparent Vd/f is about 5 L/kg. There is a wide inter-subject variability in the elemination parameters of UR-9825. The absorption rate (Cmax) and extension (AUC) of UR-9825 was dose proportional for lower-medium doses (5 to 80 mg), with a range of mean terminal half-life between 30 h and 56h. A non-liniar pharmacokinetics trend at higher doses was observed due to probalbe saturation of the metabolism process. Further studies are needed to confirm these preliminary results. P2-005. Antimicrobial properties of olive leaf D Markin, L Duek & I Berdicevsky. Technion Faculty of Medicine, P.O.B. 9649, Haifa, Israel Olive tree is amongst the most resistant, longest living plants. Olive trees can live for hundreds of years. Such a plant must therefore have significant antimicrobial properties. Research to date has focused solely on the olive fruit. Phenolic compounds isolated from olive fruits have shown to inhibit the growth of E. coli, K.pneumoniae, Staph aureus and Bacillus cereus. Oleuropein, the main active constituent inhibited growth of bacteria but not yeasts, The purpose of this study was to investigate oliveleaves regarding their antimicrobial effect upon bacteria, fungi and yeasts. We furthermore aimed to characterize the active constituent/s and determine its mode of action. Water, alcohol and ethyl acetate extracts of crude olive leaf powder, were tested to determine their antimicrobial activity. Starters of 105 microorganisms were innoculate at the appropriate extracts. Samples were taken and tested for viable counts. Water extract at 0.3 % (w/v) killed all bacteria tested (Klebsiella, E. colli, Pseudomonas, Staph aureus) following a two hour incubation of 106 bacteria whitin the plant extract. Dermatophytes were inhibited by 0.6% (w/v) plant extract and yeasts (including C.albicans) were killed following 24h incubation in a 20% (w/v) plant extract, Ethyl acetate extract inhibited bacteria at similar concentrations.Alcohol extract had no inhibitory effect. In order to determine the size of the active molecules, we dialyzed the olive leaf extract at different pore sizes. The most active fraction was found to be smaller than 7000 µr but bigger than 3500 µr. These findings suggest a therapeutic potential for olive leaves as an effective, wide acting antimicrobial substance. Based on the finding that both ethyl acetate extract and water extract of its residue showed a bacterial inhibitory effect, we are assuming that there is more than one active constituent. We are currently doing more research to characterize these substances and determine their mode of action. P2-006. Reasons for the use of antifungal agents in patients admitted to the intensive care unit Palomar M, Álvarez Lerma F, Cerda E, Jorda R, Bermejo B, and UCI (EPIFUCI) Fungal Infections Study Group, Hospital del Mar, Barcelona, Spain Introduction. To determine the use of antifungal agents in patients admittedto the Intesive Care Units (ICUs) of different hospitals in Spain and to assess patterns of prescription. Methods. Cross-sectional, multicenter, observational study. Patients admitted to the ICUs of the participating hospitals on March 23, June 22, and November 16, 1999 were enrolled in the study. In the presence of fungal isolates, fungal infection was defined by the attenting physician according to the patient’s clinical picture. Antifungal drugs were prescribed according to the physician’s criterion. Data are expressed as mean and standard deviation (SD). Results. During study period, a total of 1,468 patients were admitted to the UCIs of the 64 participating hospitals, with an average of 22.9 patients per ICU. A total of 167 (11.4%) patients were given antifungal agents. Flucanzole was administered to 61.6 % of patients followed by conventional amphotericin B in 15.5%, liposomal amphotericin B in 8.3%, and amphotericinB lipid complex in 4.9%. In 34 patients (2.3%), antifungal treatment was prescribed empirically. However, antifungal agents were administered to 133 (55.9%) of the 238 patients in whom fungal isolates were identified (infection 89.4%, colonization 25.6%).Fluconazole was the agent most frequently prescribed for the following reasons: caniduria (22.8%), persistent severe sepsis despite the use of antibiotic therapy (19.8%), isolation of fungi in samples obtained by non-invasive procedures (17.8%), fungal isolates in samples from abdominal abscesses or peritoneal exudates peroperatively (10.4%), candidemia (7.4%), and prophylactic treatment in mechanically-ventilated patients. Conclusion. Antifungal agents were used in 11.4% of ICU patients. Although fluconazole was the single agent most frequently administered, there was a large variation in the patterns of prescription of antifungal drugs.

Antifungal Drugs & Therapy

P2-007. Secondary prophylaxis for cryptococcal meningitis in HIVinfected patients treated with highly active antiretroviral therapy Pacheco P, Ferreira L, Tavares L. Hospital de Santa Maria, Lisboa, Portugal An azole based drug regimen is recommended by currently accepted guidelines for prevention of opportunistic infections in HIV-infected patients as a lifelong secondary prophylaxis, after an episode of croptococcal meningitis. There is, however, some evidence that secondary prophylaxis could be safely discontinued in patients with certain opportunistic infections (e.g. P. carinii and CMV) after some CD4+ cell count increase under highly active antiretroviral therapy (HAART). We present a microbiologically proven case of criptococcal meningitis in an HIV+, 29 year old, male IV drug abuser, successfully treated with a suppressive course of Amphotericin B three years ago. At the end of therapy induction, the CSF criptococcal antigen titier dropped from 1:1000 to 64. In spite of a daily secondary prophylaxis with fluconazole (200mg id) he was also treated with HAART (lamivudine in combination with stavudine and indinavir) for two years. Under this treatment the patient remained asymptomatic and a marked virologic response was obtained (viral load drop from 4.79 to a sustained level around 2.7 log10 HIV RNA copies/ML) as well as a sustained immunological recovery from an initial CD4+ cell count of 9 to an average of 230 CD4+ cell count/µl. The CD19+ cell counts, as marker of the B lymphocytes, remained also stable. During the last 52 weeks he stopped all treatments while resuming IV drug abuse. Now, while asymptomatic, an expected drop of CD4+ cell count to 80 and a viral load rise to 5.04log10 HIV RNA copies/ml were observed. Currently he has a normal CSF with undetectable criptococcal antigen in CSF and blood. An absence of a criptococcal disease relapse during a 52-week period, while the patient had still CD4+ cell counts at least over 80 was observed. This case and the potential drugdrug often used to treat HIV-infected patients raise the question about the real value of criptococcal meningitis secondary prophylaxis and emphasizes the importance of a multicentric collaborative study of this issue. P2-008. In vivo activity of amphotericin B, itraconazole and terbinafine in a murine model of disseminated zygomycosis Dannaoui E1, Meis JF2, Verweij PE1. 1Department of Medical Microbiology, University Medical Center St. Radboud, Nijmegen, The Netherlands; 2Departement of Medical Microbiology, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands Objectives: To evaluate the correlation between MICs determined in vitro and in vivo activity of amphotericin B (AMB), itraconazole (ITZ), and terbinafine (TER) in a murine model of disseminated zygomycosis. Methods: One isolate of Rhizopus microsporus and one isolate of Absidia corymbifera were tested. In vitro susceptibility testing was performed using an NCCLS-based microdilution technique (M38-P). Nonimmunocompromised mice were infected intravenously with spore suspensions. Treatment was started 2h after infection and continued for ten days. ITZ and TER were given by gavage twice a day at 100 mg/kg/d and 150 mg/kg/d, respectively. AMB was given at 4.5 mg/kg/d by once intraperitoneal injection. Results: The MICs of AMB, ITZ and TER were 1, 32 and 0.5 µg/ml for Rhizopus microsporus and 0.12, 0.12 and 0.25 for Absidia corymbifera, respectively. With the Rhizopus isolate, a lethal infection was obtained by day 5 to 8 for control mice receiving no active treatment. In comparision, there was 100% survival for mice receiving AMB. ITZ and TER did not prolonged survival compared to the controls. All control mice infected with Absidia died by day 8 to 9. AMB showed a good activity with 100% survival. TER did not prolonge survival compared to the controls, but ITZ was partially active with 40% survival at the end of the experiment. Conclusion: (1) AMB showed a good antifungal activity in vitro and in vivo against Rhizopus and Absidia. (2) A partial correlation between MICs and treatment outcome was seen with TER in this model. P2-009. Ventricular fibrillation under systemic therapy with amphotericin B and flucytosine - A case report A Glöckner, M Wierbitzky, T Bollmann, H Bernardt, *K Zimmermann. Clinic of Internal Medicine A; *Loeffler-Institute of Medical Microbioloy, Ernst-Moritz-Arndt University, D-17487 Greifswals, Germany A 57 year old patient was successfully treated with thrombolysis (urokinase) for pulmonary embolism due to deep venous thrombosis of lower limb. The medical history includen chronic obstructive pulmonary disease, diabetes mellitus and chronic alcoholism. Due to pneumonia on the fourth hospital day mechanical ventilation was necessary. The patient received broad spectrum antibiotics and steroids for the ongoing pulmonary infection and the severe bronchial obstruction. Microbiologic diagnostic on day 16 revealed Aspergillus fumigatus in the protected brush. Treatment with Itraconazole for 7 days was termi-

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nated due to lack of clinical improvement. Therefore a systemic therapy with (1mg/kg/d) and Flucytosine was initiated. On day 7 and 8 of the combined antimycotic treatment the patient developed repeated ventricular tachycardia (torsade de pointes) with degeneration to ventricular fibrillation. Each episode of ventricular arrhythmia was associated with hypokalaemia (3,4 and 3,9 mmol/l). There was no evidence so ever for a repeated pulmonary embolism under effective treatment with herparine (PTT 70 s; echocardiography unchanged). A subsequent performed coronary angiography revealed no severe coronary artery disease as the most common reason for cardiac events. After 3 month of treatment the patient left the hospital well stabilized for ambulant rehabilitation. In our opinion the arrhythmia was due to the proarrhythmogenic effects of Ampotericin B aggravated by the mild hypokalaemia. This case report implicates the importance of a consequent electrolyte monitoring and early substitution of potassium under systemic Amphotericin B therapy. P2-010. Efficacy of liposomal amphotericin B combined with G-CSF in a murine model of invasive infection by Scedosporium prolificans M Ortoneda1, J Capilla1, I Pujol2, FJ Pastor1, E Mayayo3, J Guarro1. 1 Universitad Rovira y Virgili, 2Hospital Universitari de Sant Joan de Reus and 3Hospital Universitari de Tarragona Joan XXIII Scedosporium prolificans is an opportunistic filamentous fungus whose clinical incidence is increasing. It is resistant to the available antifungal agents both in vivo and in vitro and most of the reported cases have been fatal irrespective of the treatment. Only two cases of systemic infection have been resolved following different therapeutic regimes. We have assayed several treatments using amphtericin B desoxycholate (AB), liposomal amphotericin B (LAB) and granulocyte colony-stimulating factors (G-CSF). Immunosuppressed Of-1 male mice were infected IV on day 0 with an inoculum of 5x104 conidia/mouse and distributed in groups of 30. The assay was based on five groups of infected animals: control group (treated with 5% dextrose from day 1 to 10), AB group (1.5 mg/kg/day of AB from day 1 to 10), LAB group (10 mg/kg/day of LAB from day 1 to 10), G-CSF group (300µg/kg/day of G-CSF from day -3 to day 5) and LAB + G-CSF group (10mg/kg/day of LAB from day 1 to 10 and 300µg/kg/day of G-CSF from day -3 to day 5). The survival data were analized with the Log-Rank test from the Kaplan-Meier method. The survival rates after the 30-day observation period were 0% for control, AB and LAB groups, 3.3% fro G-CSF group and 20% for LAB+G-CSF group. In addition, the group with combined therapy (LAB+G-CSF) showed the longest mean survival time (MTS) (13.20 days). The MTS of the other groups were 9.60 days for AB, 7.47 days for G-CSF and 7.93 days for control. Our results are very encouraging because this combined treatment has the potencial to become the therapy of choice against severe systemic infections in humans caused by this emerging fungus. Further studies evaluating higher LAB doses and variations in the G-CSF treatment will probably allow us to obtain even better results. P2-011. Low nephorotoxicity in patients receiving amphotericin B lipid complex (ABLC). A pharmacosurveillance study in Spain JM Aguado1, D Gonzalez2, on behalf of the ABLC Pharmacosurveillance Study Group. 1Hospital 12 De Octubre, Madrid, Spain; 2Laboratorios Pensa, Barcelona, Spain Background: Amphotericin B Lipid Complex (ABLC), was developed to reduce the toxicity and maximize the therapeutic utility of amphotericin B. We have conducted a multicentre prospective trial in order to evaluate the renal, haematological, hepatic and acute toxicities of this drug in Spain. Methods: 93 patients with proven or presumptive fungal fungal infections (and one with visceral leishmaniasis), received ABLC at 17 hospitals in Spain (June 1996-Dec. 1998) for the one or more reasons: 44 as first choice drug, 21 due to failur of previous systemic antifungal treatment, 21 due to amphotericin Brelated toxicity, 19 due to pretreatment renal insufficiency, and 11 patients for more than one of these reasons. Sixty percent of the patients had oncohematologic diseases. Results: Among the enrolled population, median-SD basal and final serum cratinine levels were similar (1,00-1,14mg/dL vs. 1,20-1, 1,19mg/dL, p bigger than 0,05). We did not find a correlation between the median daily dose or the median total dose of ABLC admininistered and the levels of serum creatinine at the end of the treatment (Spearmann 0,22). No increase in nephrotoxicity was detected in patients included because previous renal impairment or in those who had previously received amphotericin B. We did not detect significant changes between basal and final hemoglobin, potassium, SGPT and bilirrubin serum levels. Twenty-two patients (23,6%) developed ABLC related or presumably related adverse events. In only 12 patients a reduction or whithdrawal of ABLC was necessary. In 5 cases, the adverse event was qualified as serious, but in 3 of these 5 cases, there were alternative causes attributed

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to the adverse event. Although 23% of the patients presented fever or chills during ABLC infusion, only in one case the drug was withdrawn for this reason. Conclusion: Our study shows that ABLC is a low-nephrotoxic drug even in patients with previous renal impairment, or who had previously received amphotericin B. Adverse effects during ABLC infusion are of low intensity and can be managed with premedication. P2-012. Sporotrichoid cutaneous phaeohyphomycosis due to Alternaria infecoria in a heart transplant patient treated successfully with itraconazole Regine Horr1, Günter Marklein1, Manfred Uehrlich2, Rainer Gerdsen2, Ralf Bauer2, Sybren de Hoog3, and Klaus-Peter Schaal1. 1Institute of Medical Microbiology and Immunology, University of Bonn, 2Clinic of Dermatology, University of Bonn, Germany, 3Centraal Bureau voor Schimmelcultures, 3740 AG Baarn, The Netherlands Alternaria infectoria is a melanized fungus commonly found in decaying plant material. Cutaneous infections in humans due to Alternaria are rare and have been described mainly in immunocompromised patients. The clinical picture in these patients is variable. We report about a case of a sporotrichiod cutaneous alternariosis due to A. infectoria in a heart transplant recipient. Case report: A 61-year-old German woman, receiving oral immunosuppressive therapy in view of a history of heart transplantation two years before, developed an erythematous nodule on her left hand. Although no microorganism could be cultured from wound swab, a myotic infection was assumed and the patient was treated topically with ketoconazole. Four months later the lesions were progressive and two new lesions at the left wrist and elbow had developed. The clinical picture now resembled a sporotrichosis. Tissue samples were sent for histopathological and microbiological examination. Histology revealed a dermal granulumatous infiltrate. With Gomori silver stain hyaline septate hyphae could be seen. With Fontana Masson staining melanin was shown to be present as a dark brown pigmentation. Cultures of the tissue samples yielded growth of a dark colored mycelial fungus after 10 days of incubation. The fungus did not produce any conidia, but was identified as Alternaria infectoria by ITS rDNA sequencing. Treatment with oral itraconazole (2 x 200 mg/day) leds towards an improvement within six weeks. The therapy was continued with a daily dose of 100 mg itraconazole for five months. After this time the lesions had completely disappeared. Anamnesis revealed that the patient regularly cleans a birdhouse which might have been the reservor of the etiologic agent. P2-013. Reversion of resistance of Candida spp to fluconazole by different modulators Cidália Pina-Vaz1,2, Acácio Gonsalvez Rodrigues1,2, Sofia Costa-deOliveira1, Cristina B Tavares1, J Martinez-de-Oliveira3, A Freitas da Fonseca1. 1Department of Microbiology, Porto School of Medicine, University of Porto, 2Institute of Pathology and Molecular Immunology of Porto University, 3Department of Gynaecology, Porto School of Medicine, University of Porto, Portugal Multidrug resistance in pathogenic yeasts has been related to efflux pumps that export the drugs, therefore reducing their toxity. Some compounds (modulators) have the capacity to block these proteins. We tested 4 drugs as potential modulators fo resistance on Candida spp to fluconazole (Flu): verapamil, β-estradiol and progesterone, known inhibitors of efflus pumps on human neoplastic cells. Ibuprofen has a documented fungicidal activity at high concentrations but shows at lower doses a synergistic effect with Flu mainly on resistant strains, unrelated with the fungicidal mechanism. As it is a lipophilic compound, like other modulators, we decided to study its capacity to revert resistance by blockade of efflux pumps. Material and methods: Ten clinical isolates of Candida strains resistant to Flu and 2 sensitive were incubated with sub-inhibitory concentrations of verapamil (100 µM), β-estradiol (50 µM), progesterone (50 µM) or ibuprofen (500 µg/ml) all from Sigma. MIC values to Flu (Pfizer) associated to each modulator were determined. Yeast cells exposed and not exposed to the modulators were stained with FUN-1 (a fluorecent probe) and analyded by FC. Results: MIC values decreased 4 to 32 times when Flu is associated with the different modulators in most strains. Only C. krusei and sensitive strains did not show significant MIC variation. The FC results showed that, on strains where MIC values to Flu were reduced by the modulators, FUN-1 staining increased, exept for C. glabrata. Conclusions: Most of the resistant strains tested seem to have efflux pumps that are blocked by the compounds used, reverting the resistant phenotype. On C. krusei the mechanism of resistance seems to be different. The FUN-1 is a useful probe that can be used to detect efflux pumps. On C. glabrata the modulators blocked the Flu transport but not the FUN-1. The hormones and especially ibuprofen, associated with Flu, might be used in the clinical management of candidosis.

P2-014. Five years analysis of the Lamisil efficacy in the therapy of skin and nails mycoses Krajewska-Kulak E1, Niczyporuk W2, Lukaszuk C1. 1Department of Nursing Theory . Medical Academy in Bialystok, 2Department of Dermatology and Veneorology. Medical Academy in Bialystok, Poland The aim of this study was the prospective analysis of the Lamisil efficacy in the therapy of skin and nails mycoses after 5 years of the end therapy. 34 patients aged 31-67 years old were studied. Before five years in those patients tinea cutis glabrae was diagnosed in (15 patients), tinea unguium pedum in (13 patients) and tinea unguium manum in (6 patients). After 5 years since the end of the therapy, there was analyzed appearance, intensification or lack of clinical symptoms of mycosis and results of mycological examination and culturing of the material received from sites with previous sickness – process. On the basis clinical examination and mycological investigations of 34 patients only in one case changes in colour nails of both great toes, the presence of mild hiperceratosis and mild onycholysis and positive result of the microscope examination had found. In culturing Trichophyton rubrum (the same etiologic factor as previous) was found. After 5 years of the observation of the efficacy of lamisil therapy was following: tinea unguium pedum 92.3%, tinea unguium manuum - 100% and tinea cutis glabrae - 100%. Lamisil is effective and well tolerated drug in the therapy of mycoses of skin and nails with small rate of the relapses. Lamisil appeares efficient drug in the therapy of skin and nails mycoses. P2-015. The effect of the ELF magnetic field in comparision to antifungal drug on the laboratory animals infected by pathogenic fungi Budak A1,3, Zyss T2, Bogusz B3, Zieba E4. 1Department of Pharmaceutical Microbiology, Collegium Medicum, Jagellonian University, Poland; 2 Collegium Medicum, Jagellonian University, Kraków; 3Lab of Microbiology, Department of Analytical Diagnostics, Rydygier´s Hospital, Kraków; 4Department of Dermatology, Rydygier´s Hospital, Kraków Personal investigations, which have been carried out for 4 years, proved that the magnetic field inhibits the growth of some pathogenic dermatophytes cultured in vitro. The aim of this study was comparision of the effects of magnetic field and terbinafine on laboratory animals infected by dermatophytes. The experiments were carried out on guinea pigs, which were 2 month old, males and kept in standard conditions. The animal was infected in scarificated skin of the back. (the density of the suspension was 9 x 108 spores investigated fungal species per ml). Then it was left alone till the appearance of fungal lesion on the skin, whose diameter would be at least 1 cm. In the study species: T. mentagrophytes and M. canis were chosen. Infected animals were selected to one of the three research groups : 1/ group treated with terbinafine used superficially and to intestinal tract, 2/ group treated by magnetic field, and 3/ control group. Apparatus Magnetronic MF-10 was used to magnetic stimulation. It generated changeable magnetic field ELF (frequency of 50 Hz, daily stimulating time up to 12 hours). The period of investigation was the time from the start of the infection to healing of the animal (2-3 months). In order to estimate the effectiveness of therapy, the method of computer planimetry was applied. The area changed by illness was measured by digital camera (once a week) and it was analyzed by means of software package MultiScanBase v 8.08. We observed the inhibiting effect of magnetic field on the development of the skin lesions of some infected animals. P2-016. Antifungal effect of tetramethyldithiooxamides T Kantardjiev1, S Tabakova2, V Mircheva3. 1National Centre for Infectious and Parasitic Diseases, Sofia- Bulgaria ; 2Institute of Molecular Biology, The Bulgarian Academy of Sciences, 3Institute of Polymers, The Bulgarian Academy of Sciences, Bulgaria Tetrasubstituted dithiooxamides are a new group of antimicrobial agents. The aim of this study is to investigate the anticandidial effect of tetramethyl dithiooxamide (TMDTOA), the aseptic properties of TMDTOA treated materials and the effect of TMDTOA on ergosterol biosynthesis in yeast. Methods: MIC - microtitre broth dilution method in RPMI medium, incubation of 24 h at 35°C and inoculum size of 1x104 cfu/ml on clinical isolates of azole susceptible and azole -resistant Candida spp. Antiseptic properties:TMDTOA treated materials (rubber, gauze, leather) were plated on agar, heavily preinoculated (1x109 cfu/plate) with Candida spp., Staphylococcus aureus and Asperigillus niger alone or in mixtures. Inhibitory zones and growth on the material were determined. Effect on ergosterol biosynthesis: UV absorption spectra of crude n-haptane extracts of the non-saponifiable lipids of yeast cultures treated with subinhibitory concentrations of TMDTOA and untreated controls were compared. Results: MIC for Candida albicans (20 strains) were 0.32 to 8.0mg/l; for C. glabrata (5 strains)- .3.2-8.0 mg/l; C. krusei (3 strains)- 4-8 mg/l,

Laboratory Methods

C. parapsilosis (8 strains) - 4-16 mg/l. A sterile zone of 1-2 mm around all materails, treated with TMDTOA was observed and persisted for at least 30 days. UV absorption at the characteristic ergosterol peaks was equal for treated cultures and controls, suggesting no effect of TMDTOA on sterol biosynthesis. The significant anticandidial effect, combined with antibacterial action suggest that TMDTOA is suitable for application as an antiseptic and further studies to determine the possibilities for its developments as an antifungal drug are of interest, especialy in view with the necessity to search for novel classes of antifungals, acting independently of ergosterol biosynthesis. P2-017. Incorporation in liposomes reverts suppressive effect of amphotericin B on CD8 T cell function M Kretschmar1, G Gegina1, T Bertsch2, S Walter1, H Hof1, and T Nichterlein1. 1Institut für Medizinische Mikrobiologie und Hygiene und 2 Institut für Klinische Chemie, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany Amphotericin B is an important drug for the treatment of life-threatening fungal infections in the immunocompromised host. However, usage of the substance is severly impaired by toxic side effects, especially nephrotoxicity and modulatioin of cellular functions. This has been shown previosly also for cytotoxic T cell functions which were impaired by therapeutic doses of Amphotericin B in vitro and in a model of murine listeriosis (JID, 1999; 180: 1186-94). Most of the side effects may be ameliorated by incorporation in liposomes. Therefore, we investigated whether liposome incorporation could also revert the strong suppressive effect on the function of CD8 T cells which we have describe previously. The influence of the commercial liposomal preparation AmBisone on the cytotoxic T cell function was studied in vitro using a chromium release assay and in a model of systemic murine infection with Listeria monocytogenes and compared with the effect of commercial Amphotericin B complexed with deoxycholate (Fungizone). In vitro analysis showed that incorporation in liposomes abolished the effect of Amphotericin B on target cell lysis by peptide-specific CD8 T cell lines. Whereas there was a demontrable effect of Amphotericin B in Fungizone at a concentration of 1 mg/l, concentrations of 100 ml/l were necessary to produce a similar effect when Amphotericin B was incorporated in liposomes. For in vivo studies, mice transferred with a L. monocytogenes-specific CD8 T cell line were treated whit 0.75 mg/kg of Amphotericin B either as Fungizone or as AmBisome. When the mice were treated with AmBisone, there was a less pronounced increase of the colony-forming units in the liver in comparision with Fungizone in spite of considerably higher concentrations of Amphotericin B in this organ upon treatment with AmBisome measured with HPLC. Our data clearly demonstrate that incorporatioin of Amphotericin B into liposomes is able to reduce the inhibitory effect on protection mediated by cytotoxic T cells. P2-018. A surveillance study to evaluate the clinical safety and therapeutic efficacy of a combination therapy with flucytosine (Ancotil®) in the treatment of patients with deep seated mycosis Just-Nuebling G1, Kujath P2, Fegeler W3. 1Center of Internal Medicine/Infectiology, J.W.Goethe-University Frankfurt; 2 Medical University, Lübeck; 3Institute of Medical Microbiology, Westfaelische Wilhelms University, Muenster Background: Clinical efficacy of combination therapies with flucytosine has been demonstrated in many published studies. However, the potential toxicity of flucytosine (e.g. hematologic, hepatic, gastrointestinal) has influenced the use of this broad-spectrum antifungal drug. Unsatisfactory treatment results with antimycotic chemotherapy, esp. in severe immunocompromised patients with systemic fungal infections, have lead to an increasing interest in drug combinations. This post-marketing surveillance study was performed to reflect current patterns of medical practice in the context of treatment with flucytosine. Objectives: The aim of the study was to evaluate the clinical safety and therapeutic efficacy of a combination therapy with flucytosine in the treatment of patients with deep seated mycosis. Patients and methods: More than 200 patients with possible, very probable or proven invasive mycotic disease were enrolled in this open, prospective multicenter surveillance study. Combination partners, dosages and schedules in the treatment of deep seated mycosis were not restricted and should be determined on an individual basis in accordance with the clinical condition of the patients and the fungal species that had been identified. Blood and tissue samples, relevant clinical data and laboratory findings were recorded before and after treatment with flucytosine. Adverse events occurring during the study and their relation to the treatment with flucytosine were recorded. After the end of treatment the clinical results and success of the antifungal therapy were assessed by the investigator. Results: Most of the patients were intensive care patients after severe surgery, patients with hematological malignancies or neutropenic

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patients. The main indications for the antifungal treatment were systemic Candida infections and invasive aspergillosis. Most of the patients received a combination of amphotericin B and flucytosine. Detailed results of this study will be presented. __________________

Laboratory Methods P3-001. Evaluation of Candida ID, a new chromogenic medium for fungi isolation Fricker-Hidalgo H, Lebeau B, Pelloux H, Ambroise-Thomas P, Grillot R. Service de Parasitologie-Mycologie, Centre Hospitalier Universitaire, BP 217-38043 Grenoble, France Objective: Recently, a new plated medium Candida ID (CAID, bioMérieux) was developped to improve isolation of fungi. It allows identification of Candida albicans (blue colonies) and preidentification of others species (C.tropicalis, C. guilliermondii, C. kefyr and C. lusitaniae, pink colonies). In this study CAID was compared with Albicans ID2 (AID2) and Sabouraud gentamicin chloramphenicol agar (SGC) using 220 clinical samples. Methods: Fungi were isolated from 103 samples among 220. Growth and aspect of colonies were monitored after 24, 48, and 72 hours of culture. All colonies showing different morphologies were identified using conventional methods including agglutination tests (Bichrolatex albicans test and Krusei color, Fumouze) and the ID 32 C system (bioMerieux). Results: A total of 139 fungal strains belonging to 12 species were isolated on at least one of the three media : 125 (89.9%) on SGC, 133 (95.7%) on AID2 and 133 (95.7%) on CAID. A 98.7% sensitivity (75 among 76 Candida albicans identified by Bichrolatex albicans) and a 100% (57/57) specificity were observed in the identification of Candida albicans isolates on CAID. On AID2, 2 Candida tropicalis strains among 7 produced blue colonies with white center (specificity : 96.5%). Conclusion: This comparative study showed that CAID was able to allow the isolation of more fungus species than SGC and improved the specificity of Candida albicans identification compared to AID2. P3-002. Influence of sub-inbihitory concentrations of conventional antifungals on mitochondrial metabolism and adherence of Candida albicans to polystyrene and extracellular matrix proteins Christine Imbert, Marie-Helene Rodier and Jean-Louis Jacquemin. Laboratoire de Parasitologie et Mycologie Médicales, CHU La Milétrie, BP 577, 86021 Poitiers Cedex, France Five antifungals agents with different mechanisms of action were compared: their ability to affect mitochondrial deshydrogenase activity of Candida albicans was examined together with their ability to affect adherence of this yeast to polystyrene and extracellular matrix proteins. Amongst the antifungals tested below their corresponding MIC value, only amphotericin B inhibited mitochondrial deshydrogenase activity. Fluorocytosine and terbinafine did not affect this activity, whereas ketoconazole, itraconazole and fluconazole improved it. Furthermore, the effect of sub-MIC antifungals on adherence was dependent on the tested antifungal and the adherence surface: amphotericin B improved adherence to polystyrene and fibrinogen but inhibited adherence to extracellular matrix. On the other hand, for all the adherence surfaces tested, fluorocytosine and ketoconazole did not affect adherence, itraconazole and fluconazole increased adherence, and finally, terbinafine inhibited adherence. These data suggest that antifungals used below their corresponding MIC value can have various effects. It is therefore important to make a distinction between effects of the antifungals on the metabolism and on the adherence capacity of C. albicans. P3-003. Evaluation of PCR and Platelia Candida antigen and antibody in early detection of cantidemia in ICU patients G Dimopoulos1, A Velegraki2, A Karabinis1, NJ Legakis2. 1Dept. of Intensive Care of the District General Hospital of Athens G. Gennimatas; 2Dept. of Microbiology, Mycology Reference Labatory, Medical School , University of Athens, Greece Aim: To evaluate PCR and Platelia Candida antigen and antibody in the early diagnosis of canidemia in intesive care unit patients. Methods: Over 12 months (December 198-99) among 137 ICU admissions, 34 (25%) patients with candiduria, which appeared after 6±3 days after their hospitalization, were clinically and mycologically investigated. The mean age of the patients was 52±8 years, the mean duration of the mechanical ventilation 11.7 days, the mean length of ICU stay 15.6 days and the APACHE II Score was 19±6. Upon laboratory confirmation of candiduria, whole blood samples were tested for Candida DNA sequences with multiple PCR reactions every 7 days during their ICU