F 6

EFFICACY OF AMPHOTERICIN B COLLOIDAL DISPERSION IN THE TREATMENT OF SYSTEMIC CANDIDA INFECTIONS

G. Farkas, M. Kakulya, L. Leindler, E. Nagy
University of Szeged, Faculty of Medicine, Department of Surgery and Institute of Clinical Mirobiology, Szeged, Hungary

The incidence of fungal infections has increased during recent decades. These fungal infections have become common in surgical patients, mainly after major operations or reoperations with prolonged hospitalization. Thus, the rate of systemic infections due to Candida species also has increased greatly in recent years with high mortality. Amphotericin B colloidal dispersion (ABCD) was developed to reduce the nephrotoxicity associated with conventional Amphotericin B. This review presents the clinical results of ABCD therapy in patients with systemic fluconazole-resistant Candida infection.

Methods: In the past 2 years, 10 patients (f/m ratio: 2/8; mean age: 43.5 years) with infected pancreatic necrosis were identified as being infected with systemic Candida species. All these cases involved mixed bacterial and fungal infections. Candida was isolated from the blood stream, an intra-abdominal discharge, deep tissues, urine or sputum. All Candida species (albicans: 7; glabrata: 3; krusei: 2) were fluconazole resistant. After the diagnosis of systemic fungal infection, all the patients were treated with 4 mg/kg ABCD medication daily.

Results: No side-effects of ABCD were noted in any of our 10 patients. The kidney and liver functions were normal in 9 patients; the kidney function in one patient with moderately elevated creatinine and UN levels did not deteriorate, and this patient did not become uraemic. There were 2 deaths following pulmonary embolism or severe bacterial sepsis, but 8 patients were successfully treated.

Conclusion: Our results clearly demonstrate that Amphotericin B colloidal dispersion is sufficient and safe treatment for systemic Candida infections, and severe side-effects do not occur.

F 7

FUNGAL COLONIZATION IN NEUTROPENIC PATIENTS: A RANDOMIZED STUDY COMPARING ITRACONAZOLE SOLUTION AND AMPHOTERICIN B SOLUTION

C. Lass-Flörl, E. Gunsilius, G. Gastl, H. Ulmer, A. Petzer
Institut für Hygiene, Department für Hämatologie, Innere Medizin, Universität Innsbruck, Austria

In this study we assessed the impact of prophylaxis with the oral itraconazole solution and amphotericin B solution on fungal colonization and infection in a randomized study among patients with haematological malignancies and neutropenia. Infecting and colonizing Candida strains were genotyped by random amplification of polymorphic DNA (RAPD) analysis.
106 patients were evaluated in this study, 52 patients in the itraconazole and 54 in the amphotericin B arm. During neutropenia fungal colonization in oropharynx occurred in 11 (19.6%) and 24 (40.6%), in rectum occurred in 11 (19.6%) and 23 (38.9%) courses in the itraconazole and amphotericin B group (P < 0.05), respectively. Candida albicans was the most prevalent species in both study groups. The occurrence of invasive candidas was significantly increased in multicolonized compared to monocolonized patients. In the amphotericin B group 20 and in the itraconazole group 2 neutropenic patients showed multicolonization with Candida spp. (P < 0.05). Overall fungal infections were 3.8% in the itraconazole and 14.8% in the amphotericin B group (P < 0.05). RAPD typing showed oropharynx strains involved in superficial infections in 4 of 5 patients. In all 4 patients with deep fungal infections it appears that the colonizing rectum strains were identical to infecting strains of Candida spp.
Itraconazole solution reduced significantly Candida colonization and infection compared to amphotericin B solution. Most patients remained colonized with the same strains for the entire study period, irrespective of antifungal prophylaxis.

F 8

RESULTS OF A PROSPECTIVE COMPARATIVE TRIAL EVALUATION TWO POLYMERASE CHAIN REACTION ASSAYS AND A GALACTOMANNAN ELISA TO DIAGNOSE INVASIVE ASPERGILLOSIS IN HAEMATOLOGIC HIGH RISK PATIENTS

M. Hummel, D. Schleiermacher, B. Spiess, H. Skladny, H. Mörz, R. Hehlmann,
D. Buchheidt
III. Medizinische Klinik, Klinikum Mannheim, Universität Heidelberg, Mannheim, Germany

Early diagnosis of invasive aspergillosis (IA) is still difficult to achieve. Therefore we prospectively evaluated a nested polymerase chain reaction (PCR) assay, a LightCycler™ mediated assay, and a galactomannan ELISA (GM) in neutropenic patients with haematologic malignancies at high risk for IA to validate their significance in diagnosing IA. During 205 treatment episodes in 165 patients from six centres, a nested PCR assay and GM testing were performed at regular intervals. Positive PCR results were quantified by a LightCycler™ mediated assay. Patients episodes were stratified according to EORTC/MSG 2002 criteria. PCR and serology results were correlated with the diagnostic classification. 104 (50.7%) patients episodes had no evidence of IA, 8 (3.9%) had proven, 3 (1.5%) had probable and 90 (43.9%) had possible IA. Overall, there were 119 nested PCR assay positive and 1408 negative samples. Sensitivity rates for the nested PCR assay were up to 63.6%, specificity rates ranged between 63.5% and 93.5%. Sensitivity and specificity rates for GM were 14.3% and 98.9%, respectively. The LightCycler™ PCR assay yielded positive results in 21.4%. These results support the concept of DNAaemia occurring transiently and at a low level. In patients at high risk for IA, positive Aspergillus PCR results are highly suggestive for IA. Our data show distinctly higher sensitivity rates for the nested PCR assay as compared to GM testing and a species specific quantitative PCR assay.

Supported by a grant of the “Deutsche Jose Carreras Leukämie-Stiftung” (DJCLS-R00/07).

F 9

THE IMPACT OF MULTIPLE RESISTANT S. HAEMOLYTICUS IN NICU-ACQUIRED INFECTION

K. Kristóf, S. Kardos, V. Cser, A. Ghidan, F. Rozgonyi
Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary

Objective: Coagulase-negative staphylococci are now one of the major causes of bacteriaemia in neonatal intensive care units. The aims of this study have been to quantify the impact of S. haemolyticus in NICU-acquired infections occurred between January 2000 and March 2003.

Methods: S. haemolyticus isolates were taxonomically identified with traditional methods, and confirmations were made in the API or ATB system (bioMérieux). Antibiotic sensitivities were determined with the disc diffusion test and with the microdilution method according to the NCCLS guideline. Genetic backgrounds of the oxacillin resistance of staphylococci were confirmed by PCR techniques.

Results: 640 coagulase-negative staphylococci were isolated from different samples of the neonates cared at the three NICU of the university hospitals. 95 (15%) isolates were S. haemolyticus among these. Each strain had the mecA gene, therefore was resistant to oxacillin. Most of the strains were resistant to macrolides (90%), gentamicin (98%), tobramycin (96%) and ciprofloxacin (64%). Amikacin and netilmicin were the most effective among aminoglycosides with a sensitivity rate of 41.1 % and 56.6% of the staphylococci, respectively. There was found no resistance strain to vancomycin, but minimum inhibitory concentration (MIC) levels of the other glycopeptides-teicoplanin were elevated. The geometric mean of the MIC of vancomycin and teicoplanin were 2.3 mg/ml and 13 mg/ml, respectively.

Conclusion: The multiple resistant S. haemolyticus strains are becoming more frequent gram-positive causative agents of NICU-aquired infections because of their extraordinary ability to adapt and tolerate the environmental antibiotic pressures.

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Problem Organisms in the Swiss External Quality Control Scheme for Bacteriology/Mycology, 2002

PROBLEM ORGANISMS IN THE SWISS EXTERNAL QUALITY CONTROL SCHEME FOR BACTERIOLOGY/MYCOLOGY, 2002

A. von Graevenitz
Med. Mikrobiologie, Universität Zürich, Schweiz

Among the 12 samples sent out in 2002 to our approx. 100 participants, there were three (each divided up into 2 sub-samples with different contents) whose average grade was < 75% and thus deemed unsatisfactory.

CDC groups EF-4a and EF-4b from a cat bite were diagnosed only by 21/45 and 13/45 participants, respectively, probably due to absence of these organisms from the data base of most commercial identification galleries. The most frequent misdiagnoses were Pasteurella, Capnocytophaga, Moraxella, and Neisseria spp.

Mixed cultures from deep wounds containing Enterobacter gergoviae plus Fusobacterium mortiferum or Hafnia alvei plus Bacteroides fragilis were diagnosed as monocultures containing the enterobacterial species only by 29/47 and 15/47 participants, respectively, due to lack of proper anaerobic techniques and/or media.

Finally, even the mere genus diagnoses of Nocardia asteroides and Mycobacterium fortuitum from an abscess were missed by 10/41 and 12/41 participants, respectively, due to use of commercial galleries whose data base did not contain the organisms, and/or due to failure to use acid-fast stains.

Reasons for failures to diagnose the above bacteria will be discussed.

F 11

EMERGENCE OF QUINOLONE RESISTANT NEISSERIA GONORRHOEAE IN PAKISTAN

K. Saleh
Aga Khan University, Pakistan

Objective: The objective of this study is to report emergence and trend of QRNG from a tertiary care hospital laboratory in Pakistan.

Methods: This study was conducted in a tertiary care hospital in Karachi, Pakistan between 1992-2002. Laboratory based surveillance data was retrieved. During this period cultures for Neisseria gonorrhoeae were plated on Gonococcus agar. Identification was by standard biochemical tests. Antimicrobial susceptibility testing was performed on gonococcus agar using Kirby Bauer technique. Ofloxacin MICs were assessed on isolates received in 2002 using E test strips. Clinical information was obtained on patients with ofloxacin MIC more than 2 mg/mL. Data analysis was by SPSS version 10.

Results: During 1992-2002, 270 isolates of N. gonorrhoeae were isolated. QRNG was first isolated in 1999. Since then 33 isolates of QRNG have been identified. In 2002, ofloxacin MICs were performed on 12 isolates found to be resistant by Kirby Bauer technique. All of these QRNG isolates showed high level of resistance (MIC > 4 µg/mL). There was treatment failure in all of patients with QRNG isolates receiving quinolone. Antimicrobial susceptibilities to Penicillin and Tetracycline showed marked variations over the past 10 years. However, none of the isolates has shown resistance to Ceftriaxone.

Conclusion: We therefore, in this first report of QRNG isolates from Pakistan, report an increasing trend of QRNG isolates as seen in our laboratory with an associated increase in resistance to penicillin and tetracycline. We strongly urge the need for detection and documentation of the resistant gonococcal isolates at a national level.

F 12

EFFECTS OF DIFFERENT ANTIBIOTICS ON PERIODONTOPATHOGENIC BACTERIA IN BIOFILM

W. Pfister, T. Seltmann, S. Eick
Institute of Medical Microbiology, University of Jena, Germany

Biofilms are barriers to antibiotics. Higher concentrations than the MIC-values to planctonic bacteria are necessary to reduce bacterial growth in biofilms. Our study intended to examine this statement with antibiotics frequently used in periodontics. We investigated clindamycin, doxycyclin, moxifloxacin, and metronidazole and the species Porphyromonas gingivalis ATCC 33277, Actinobacillus actinomycetemcomitans Y4, and Streptococcus constellatus J384b (clinical isolate). Concentrations of the antibiotics tested were the MIC, and the 5, 10, 50 and 100fold MICs. The MIC of the antibiotics to the planctonic species were determined by E test. Then slides were covered with synthetic saliva supplemented with 5% albumine. They were transferred into bacterial suspensions (broth with 10 6 /mL microorganisms) and were allowed to stay here for 24 hours at 37°C. After incubation the slides were transferred into antibiotic solutions with concentrations mentioned above and into PBS without antibiotic as control. After 0, 2, 4, 8, 24 and 48 h bacteria were removed by sonication and the cfu were determined. Clindamycin reduced the growth of P. gingivalis in biofilm only by 50fold MIC. Doxycyclin reduced the growth of A. actinomycetemcomitans in concentrations >10 MICs. P. gingivalis was eliminated by 50fold MIC. Moxifloxacin showed a reduction of all species tested and an elimination of all bacteria in concentrations > 10fold MIC. Metronidazole was not able to eliminate P. gingivalis. So efficacy of antibiotics to bacteria in biofilm was different. But considering the MIC-values and achievable effective levels it has to be stated that elimination of bacteria in biofilm cannot be reached by antibiotics alone.

F 12a

DETECTION AND IDENTIFICATION OF FUNGI FROM FUNGUS BALLS OF THE MAXILLARY SINUS BY MOLECULAR TECHNIQUES

B. Willinger, A. Obradovic, B. Selitsch, J. Beck-Mannagetta, W. Buzina, H. Braun,
P. Apfalter, A.M. Hirschl, A. Makristathis, M. Rotter
Division of Clinical Microbiology, Institute of Hygiene and Medical Microbiology, University of Vienna, Austria
Dept. of Oral and Maxillo-Facial Surgery, Landeskrankenhaus Salzburg, Austria
ENT University Hospital, Karl-Franzens-University Graz, Austria

The aim of this study was to find a reliable method for the detection and identification of fungi in fungus balls of the maxillary sinus, and to evaluate the spectrum of fungi in these samples. 112 samples were obtained from patients with histologically proven fungal infections; 81 samples were paraffin-embedded tissue sections of the maxillary sinus. In 31 cases, sinus contents without paraffin embedding were sent for investigation. PCR amplification with universal fungal primers for 28S-rDNA and amplicon identification by hybridization with species-specific probes for Aspergillus fumigatus, A. flavus, A. niger, A. terreus, A. glaucus, Pseudallescheria boydii, Candida albicans, and C. glabrata were performed for all samples. Furthermore, PCR products were sequenced. Fresh samples were also cultivated. Fungal DNA was detected in all of the fresh samples, but only in 71 paraffin-embedded tissue samples (87.7 %). Sequence analysis was the most sensitive technique, as results could be obtained for 28 (90.3 %) fresh samples by this method in comparison to 24 (77.4 %) samples by hybridization and 16 (51.6 %) samples by culture. However, sequence analysis delivered a result only for 36 (50.7 %) of the paraffin-embedded specimens. Hybridization showed reliable results for A. fumigatus, which proved to be the most common agent in fungus balls of the maxillary sinus. Other Aspergillus species and other genera were rarely found.