key: cord-0038039-s37255t0
authors: nan
title: Abstracts cont.
date: 2015-12-28
journal: Clin Microbiol Infect
DOI: 10.1111/j.1469-0691.2005.01135.x
sha: 9166de57df00f5c7d7c0517594350b8fcd3c916c
doc_id: 38039
cord_uid: s37255t0

nan

Health care issues, public health, pharmaco-economics R1949 Malaria problem in Afghanistan: malaria scanning results of the Turkish medical aid group after the war Y.A. Ö ner, S.E. Okutan, E. Artinyan, B. Kocazeybek (Istanbul, TR)

Objectives: Malaria is a parasitic infection caused by Plasmodium species and it is especially seen in tropical and subtropical areas. We aimed to evaluate the effects of the infection in Afghanistan, which is an endemic place for malaria and had severe socio-economical lost after the war. We also compared these data with the ones that were recorded before the war. Methods: Blood samples were taken from 376 malaria suspected patients who come to the health centre, established by the medical group of Istanbul Medical Faculty in 2002, Afghanistan. Blood samples were screened using the OPTIMAL Rapid Malaria Test and Giemsa staining method. Results: In 95 (25.3%) patients diagnosis was malaria. In 65 patients (17.3%) the agent of the infection was P. falciparum and in 30 patients (8%) agents were other Plasmodium species. Conclusions: Reported data of malaria in Afghanistan are limited by the only studies carried on Afghan refugees in bordering countries. In this study given data were collected from the present Afghan people living in their home country even after the war. We emphasized the increasing importance of the infection in Afghanistan where malaria is endemic and there is severe socio-economic damage after the war.

Prevalence of Salmonella carriers among food handlers in Hamadan, western Iran R. Yousefi Mashouf, M. Ranjbar (Hamadan, IR)

Objectives: Salmonella is one of the major causes of human diseases related to food consumption. Some infected individuals recovering from this infection become temporary or permanent carriers, harbouring the organisms in the gallbladder, biliary tract, or rarely in their intestine. The objective of this study was prevalence of S. typhi and S. para-typhi carriers among food handlers. Method: In this study, 210 persons who were working in supplying and distribution of the food-stuffs were investigated. The fecal samples from food handlers were collected and cultured on a suitable media. The organisms were identified by relevant antisera. The data was gathered through questionnaire and analysed using EPI6 statistical software. Results: Of 210 stool cultures, 5 cases (2.38%) were positive for Salmonella species. Two (0.94%) were S. typhi, two (0.94%) S. para-typhi B, and one (0.47%) Salmonella non-typhi. Positive cases were tested again after one month and one of them (non-typhi) was negative in culture. So, the rate of infection was 0.19%. Conclusion: Our results showed that prevalence of S. typhi and S. para-typhi carriers among food handlers in Hamadan was approximately similar to those in the third world countries. Since food handler carriers are at high risk for transition of organisms, which involved in foodborne diseases, they must be checked regularly for health.

Seroprevalence of Haemophilus influenzae Type B in children aged 1-5 years old in the region of a primary health care unit in Antalya, Turkey Objectives: Aim of this study was to determine the natural immunity to Haemophilus influenzae type b (Hib) and contributed factors in children because there is limited data about the seroprevalence of Hib in Turkey. Methods: Presented study was performed in healthy children aged 13-60 months living in the region of Primary Health Care Unit Number 17, Antalya. A total of 378 children were selected for sampling. No child had been vaccinated before against Hib. Probable factors were asked to parents of the children affecting Hib natural immunity. Questionnaire forms were filled by us. Blood samples were taken from those children. Capsular polysaccharide specific IgG antibody (anti-PRP) levels were determined in the sera by EIA methods. 0.15 microgram/ml is accepted as positive limit level of anti-PRP IgG antibody. Dependent and independent variables were evaluated by statistical methods. Results: Antibody levels were below 0.15 l/ml in 10 (2.6%) and antibody levels were above 0.15 l/ml in 368 (97.4%) of children in the study group. No significant relationships were found geometric mean titres of antibodies with year's intervals (p > 0.05). Relationships were established between the Hib natural immunity and frequency of upper respiratory tract infection and bronchopneumonia (p < 0.05). No significant relationships were found between the natural immunity and age, gender, breastfeeding, socioeconomic level, attendance day care center, number of siblings, exposure to otitis media and sinusitis (p > 0.05). Conclusion: In our study we determined the natural immunity rate was high even in very early ages in Antalya. This fact should be evaluated by nation wide surveillance programme of invasive Hib diseases and nasopharyngeal colonisation rate before making a decision for Hib routine vaccination in Turkey.

Determination of the quantity of Aflatoxin M1

contamination in traditional and UF cheese in Shiraz, Iran S. Alborzi, B. Pourabbas, M.A. Hanifpour, M. Rashidi (Shiraz, IR) Objectives: Aflatoxins are toxic mold metabolites produced by toxigenic strains of Aspergillus species. They have an important role in the occurrence of some human diseases such as liver cancer, chronic hepatitis and cirrhosis. When animals eat foodstuffs containing aflatoxin B1, these toxins will be metabolized and excreted as aflatoxin M1 in their milk. A aflatoxin M1 is resistant to thermal inactivation and is not destroyed completely by pasteurization, autoclaving or the other food processing procedures. The aim of this study was to evaluate the quantity of aflatoxin M1 contamination in UF and traditional cheeses in comparison with the primary milk contamination in Shiraz city (Iran). Methods: Forty-eight UF cheese, 48 traditional cheese and 48 milk samples which were used to produce those cheeses were collected from one of the cheese factories in Shiraz. The amount of aflatoxin M1 in these samples was determined by ELISA test Using Ridascreen Aflatoxin M1 kit (r-biopharm, Germany). Aflatoxins are Soluble in polar compounds so water phase of centrifuged milk samples and methanol phase of cheese samples, which were prepared with solvents such as methanol, PBS buffer and heptan were used to determine the quantity of aflatoxin M1. Results: Thirty per cent of milk samples had contamination more than maximum tolerated level (50 ng/kg). The range of aflatoxin M1 contamination in UF cheese samples was 27-137 ng/kg and that of traditional cheese samples was 25-88 ng/kg. So none of the UF and traditional cheese samples have contamination more than maximum tolerated level, which has been accepted by European countries (250 ng/kg). This study showed that in transforming the milk to UF and traditional Cheeses 61.9% and 77.1% of aflotoxins were eradicated in whey, respectively while 38.1% and 22.9% of aflatoxins remained respectively, which shows no statistical differences between UF and traditional cheeses in contamination with aflatoxins. Conclusion: Although 30% of pasteurized milk had contamination more than maximum tolerated level with aflatoxin M1, none of the cheeses, which produced from those pasteurized milk have contamination more than maximum tolerated level. We concluded that as aflatoxins are soluble in polar compounds, most of them will be gone out with whey and little of them will remained in cheeses.

Evaluation of awareness of healthcare workers about standard health precaution and isolation M. Jamshidi, A. Jamshidi (Bandar Abbas, IR)

Objective: To evaluate knowledge of health care workers about health precaution and its association. Methods: This descriptive study is done on 191 health care workers, working in teaching hospitals of Hormozgan university of medical sciences in southern Iran. Data is collected by giving a question are including questions about knowledge of healthcare workers (HCW) about standard preventing health precaution and demographic data. The results from questionnaires is analysed by EPI INFO2000 program. For finding association we used Fisher test and chi-square. We classified HCW according to scores that they take into four groups; a (good), b (moderate), c (weak), d (very weak). Results: One hundred and ninety-one health care workers participate in this program. 85.5% was female with average age of 31.8 ± 5.6 .average work experience was 8.4 ± 6 years. 4.2% of health care workers was in group a, 57.6% in group b, 35.1%in group c and 3.1% in group d. There was no statistically significant correlation between scores and age, sex or work experience but there was significant correlation between different hospitals and different wards. High scores was seen in paediatric hospital and in ICU, CCU and emergency ward. Conclusion: Results of this study showed that awareness of healthcare workers about preventing precaution is not enough in this university and educational program in this field is necessary. Crimean-Congo Haemorrhagic Fever is an Arboviral Zoonotic infection which several cases of that have been reported in Iran. Measurement of antibody level of Crimean-Congo Haemorrhagic Fever among local and imported sheep's in Isfahan province and presence of tick on their body are the main purposes of this study. This cross sectional study has been performed among 372 local and the same amount of imported sheep's, regarding to the presence of IgG antibody of Crimean-Congo Haemorrhagic Fever and tick on their body. The study was made with the special helps of Arbovirus laboratory of Iranian Pasteur Institute on 2002. 286 (76.9%) of local and 223 (57.8%) of imported sheep's have had seropositive results but these data were statistically meaningless (P = 0.094). Ticks have been found on the body surface of 115 (31%) of local sheep's but among imported one's were totally absent. The results of this study revealed the endemic spreading of CCHF in the sheep's in Isfahan province and it needs special attention to prevent the infection in the communities and occupational exposure. Objectives: Role of Helicobacter pylori (HP) in many gastrointestinal disorders such as peptic ulcer , gastritis, intestinal metaplasia and stomach cancer is established. In order to determine risk factors of its infection this study was done. Methods: Two hundred and thirty-two patients with gastrointestinal signs were studied. Endoscopy was done by internist and 2-3 biopsies from antrum was taken. Specimens were processed by Shandon tissue Processor (Citadel). Prepared slides were stained by Hematoxycillin and Eosin & also giemsa. Two pathologists and one microbiologist studied the slides. Patients, blood groups were determined. Patients, data including age, sex, smoking, residential status, clinical signs, pathologic diagnosis, blood group were recorded and then analysed. Results: Of 232 patients 130 (56%) were females and 102 (44%) were males. 222 patients (124 women and 96 men) showed HP infection . Frequency of infection in females was 95.4% and in males 94%. 88 patients (38%) were in age group £30 and 144 (62%) in age group 31-80 years. HP was detected in 84 (95.4%) in first group and 136 (94.4% ) in second, group. 95.7% rural and 41.8% urban showed HP infection. The most frequent blood group was B (78%). All smoked patients and patients with history of smoking showed infection. HP infection in active gastritis was 98.3% and inactive gastritis with intestinal metaplasia was 64.2%. The most common symptom was epigastric pain and the least common was hiccup and vomiting. Conclusions: Frequency of HP infection is high (95.7%). No relation is between age , sex, clinical signs and HP infection. Persons with blood group B are more predipose to HP and smokers are high risk for HP infection. No difference is between urban and rural.

The study of prevalence of aetiology and outcome of septic shock patients admitted at obstetric and gynecologic department Z. Shahshahan, S. Boroomand (Isfahan, IR)

Objectives: The incidence of sepsis, the precursor of septic shock, has continued to increase over past decades. The aim of this study was to evaluate the outcome and aetiology of septic shock in Iranian patients. Methods: During the years 1992-2002 , we examined the blood, urine, uterus discharge culture of patients who were known cases of septic shock and the outcome and aetiology of the patients were recorded. Results: Four hundred patients with a mean age of 30 and a range of 25-35 were included. The prevalence of aetiology was 299 (74%) septic abortion, 55 (14%) endometritis, 27 (7%) pyelonephritis, and 10 (2.5%) chorioamnionitis, and 172 (43%) died. The results of culture were: E. coli (50%) klebsiella (20%), enterobacter SP (15%) streptococcus SP (10.5%) and others (4.5%). Conclusion: According to this study illegal abortion was the most common cause of septic shock, therefore education effective methods of contraception are recommended to women.

Co-infection of Salmonella typhi, Salmonella paratyphi, and Giardia lamblis after travelling abroad Introduction: We experienced a case of co-infection of Salmonella typhi, Salmonella paratyphi, and Giardia lamblis after traveling abroad. Case: A Korean male who was 23 year old had traveled India and Thailand for two weeks on January, 2004. He had a first febrile sense on 10 days later after traveling and started watery diarrhoea over 10 times in a day for a week. Just after he returned to Republic of Korea, he visited outpatient clinic of Division of Infectious Diseases in our hospital. He had been drinking water in bottles which were not completely corked and foods which were sold on the side of the streets in India and Thailand. He was acute ill looking and patient's body temperature was 40°C, pulse rate 78/min, respiration rate 24/min, and blood pressure 120/ 70 mmHg. Physical examinations were shown that very dehydrated tongue and decreased skin turgor but no other remarkable findings. Laboratory findings were; in CBC, WBC 3,450/lL (neutrophil 67.8%, lymphocyte 18.2%, monocyte 13.8%), hemoglobin 10.9 g/dl, platelet 143,400/lL; in chemistry, AST 106 IU/L, ALT 71 IU/L, albumin 3.0 g/dl, total cholesterol 66 mg/dl; peripheral blood smear for malaria was negative; stool occult blood positive. We examined stool wet smear by microcopy and discover the trophozoites of Giardia lamblis. We prescribed metronidazole 500 mg tid po but patient's fever was not decreased after three days later. At forth hospital day, Salmonella typhi was isolated from blood culture. So, we added ciprofloxacin 500 mg bid po and then patient's body temperature normalized after four days later. Each antibiotic administered for 10 days and then he discharged hospital after results of follow-up stool culture were negative. But, three days later, he had high fever and was hospitalized again. Salmonella paratyphi were isolated from his blood cultures. It was a question why Salmonella paratyphi had not been isolated from previous blood and stool cultures and had not eradicated despite administer of ciprofloxacin. There were no outbreaks of Salmonella paratyphi in our hospital and his community. He had not eaten out during admission and after discharge. We prescribed ciprofloxacin 500 mg bid po again. After 10 days later, he was cured and had no recurred. Conclusion: Although we do not know the correct cause for late onset of Salmonella paratyphi bacteraemia, we suggest strongly that the co-infection of three pathogens was caused by foodborne infection during his traveling. Objectives: Ceftobiprole (BPR), formerly BAL9141, is the first of a new class of cephalosporins and has broad-spectrum activity towards a wide range of Gram-negative and Gram-positive hospital-acquired pathogens including MRSA. Pseudomonas aeruginosa (PA) is a common nosocomial pathogen, which has developed high resistance rates to various antibiotics. In this study we investigated the in vitro activity of BPR and other commonly used anti-pseudomonal antibiotics against a collection of clinical P. aeruginosa isolates. Methods: A total of 407 organisms originating from hospitals in Europe (Belgium, France, Germany, Italy, and Spain) (n = 204) and throughout the USA (n = 203), were studied. All isolates were non-repeat isolates from hospitalized patients during 2001-4. MICs of BPR, ceftazidime, ciprofloxacin, imipenem, piperacillin/tazobactam, and tobramycin were determined by broth microdilution according to NCCLS guidelines. BPR breakpoints used were 4 mg/L susceptible, 8 mg/L intermediate, and 16 mg/L resistant. Results: Similar other recent reports, 14.2 and 9.4% of isolates were imipenem resistant in EU and USA, respectively. At 8 mg/ L, 71.6/78.3% EU and US, respectively PA isolates were inhibited by BPR, compared to 76.5/79.8% for ceftazidime. The two drugs exhibited a high degree of cross-resistance.

Conclusion: In addition to its anti-MRSA activity, the spectrum of activity of BPR towards P. aeruginosa resembles that of ceftazidime.

Pharmacokinetics, pharmacodynamics, drug interactions, tolerability R1959 Importance of the infecting organism in antibiotic penetration into the middle ear in experimental otitis media Objective: To determine the rate of antibiotic penetration into the ME in experimental acute otitis media caused by Streptococcus pneumoniae (Sp), Haemophilus influenzae (Hi), or a combination of both (Sp + Hi). Methods: A gerbil model of otitis media caused by either Sp, Hi, and Sp plus Hi was induced. Animals were sbc treated with amoxicillin (AMX), cefuroxime (CXM) or erythromycin (ERY) 2, 10, and 18 h after bacterial challenge and antibiotic efficacy was evaluated clinically and bacteriologically. Antibiotic concentrations were determined in the serum of healthy animals as well as in the middle ear 90 min after drug administration in infected animals. Results: The percentage of drug penetration into the middle ear in animals with otitis media in relation to the inoculated organism was as follows:

Conclusions: 1) The rate of penetration into the middle ear for betalactam antibiotics ranged approximately from 20 to 30% when animals were infected with Sp (alone or combined with Hi) but such a rate was approximately 8 to 10% when the organism involved was Hi. 2) The erythromycin rate penetration ranged approximately from 40 to 50% whatever it was the microorganism involved.

Penetration of telithromycin into nasal mucosa and ethmoid bone of patients T.S. Kuehnel, C. Schurr, K. Lotter, F. Kees (Regensburg, D) Objectives: The ketolide telithromycin has a focused spectrum of activity against respiratory tract pathogens, including penicillin-and erythromycin-resistant pneumococci, as well as intracellular and atypical bacteria. The aim of the present study was to investigate the penetration of telithromycin into nasal mucosa and ethmoid bone of patients to provide kinetic data. Methods: The study was designed as an exploratory population kinetics trial. A total of 29 patients undergoing rhinosurgery for chronic sinusitis were evaluated. The patients received a single oral dose of telithromycin 800 mg. Samples of blood, nasal mucus, nasal mucosa and ethmoid bone were collected during surgery 3, 6, 9, 15 and 24 h post-drug administration. Drug concentrations were determined by HPLC with fluorimetric detection. Results: The telithromycin mean (SD) concentrations 3-24 h after administration are listed in the Table. The achieved concentrations were generally above the MIC90 of common pathogens in upper respiratory tract infections (S. pneumoniae 0.12 mg/L, S. aureus [MSSA] 0.06 mg/L, M. catarrhalis 0.12 mg/ L; data from the PROTEKT surveillance study). The relative Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 systemic exposure, expressed by the ratio of the area under the concentration-time curve in tissue vs plasma, was 1.0 for nasal mucus, 5.9 for nasal mucosa and 1.6 for ethmoid bone, respectively.

The chinolones significantly increased the release of verotoxin2 from clinical VTEC O26 in vitro Objectives: Verotoxin-producing Escherichia coli (VTEC) cause hemolytic-uremic syndrome (HUS) mostly in children. Children hospitalised at Intensive Care Units are given antibiotics prophylaxis. However, it has been recognized that certain antimicrobial drugs have capacity to enhance the release of verotoxin. We have investigated the influence of antimicrobial drugs used against gram-negative bacteria to verotoxin2-positive clinical VTEC O26 in vitro.

The clinical VTEC strain isolated from 4-year-old child suffering from HUS was incubated with ampicillin/ sulbactam, meropenem, cefepime, piperaciline, piperacilline/ tazobactam, cefotaxime, ceftazidime, gentamicine, amikacin, ciprofloxacine and trimethoprime/sulfamethoxazole using dilution method. After 2 h and 4 h incubation, the verotoxin was separated and the presence of verotoxin was confirmed by ELISA with monoclonal antibodies against verotoxin2. We used t-test for statistical analysis.

Results: Based on ELISA tests, we have not found any significant increase or decrease of verotoxin release after 2 h incubation. However, after 4 h incubation we have found that ciprofloxacin (p < 0.05) significantly induced the production of verotoxin 2 and verotoxin release from clinical VTEC strain was significantly decreased by meropenem and amikacine (p < 0.05) compared with clinical VTEC strain incubated without antibiotics.

Conclusions: Antimicrobial drugs play role not only as inhibitors of the bacterial growth but some of them can activate the production of important virulence factors. Therefore, it is important not only isolation of VTEC from HUS patients but also detection of the influence of antibiotics used for prophylaxis in hospitals to verotoxin production of isolated strain.

Continuous versus intermittent intravenous administration of antibiotics: a meta-analysis of randomised controlled trials S. Kasiakou, G. Sermaides, A. Michalopoulos, E. Soteriades, M. Falagas (Athens, GR; Boston, USA)

Background: Intermittent intravenous administration of antibiotics is the first line approach in the management of severe infections worldwide. However, the potential benefits of alternative modes of administration of antibiotics, including the continuous intravenous infusion, deserve further evaluation. We performed a meta-analysis of randomized controlled trials (RCTs) to compare the effectiveness and toxicity of the continuous versus the intermittent mode of intravenous administration of various antibiotics. Methods: Data for this meta-analysis were identified from PubMed (01/1950 to 09/2004), Current Contents, Cochrane central register of controlled trials, and references from relevant articles. RCTs comparing the continuous with the intermittent intravenous administration of the same antibiotic regimen, and examining effectiveness, mortality and/or toxicity were included in the analysis. Experimental trials, studies focusing on pharmacokinetic and/or pharmacodynamic parameters, trials concerning antifungal, antiviral, or antiparasitic agents, and surgical prophylaxis trials were excluded. We used data conforming to standard outcome definitions of clinical failure and nephrotoxicity.

Results: A total of 20 comparisons from 9 randomized controlled trials (RCTs) studying a-lactams (6), aminoglycosides (2) , and vancomycin (1) were included in the meta-analysis. Publication bias and statistically significant heterogeneity was not observed among the analysed studies. A better outcome was observed with the continuous intravenous administration of antibiotics; the pooled odds ratio for clinical failure (10 comparisons from 7 RCTs) was 0.73 (95% CI, 0.53-1.01) for the continuous compared to the intermittent intravenous administration. Regarding mortality (5 comparisons from 5 RCTs) and nephrotoxicity (5 comparisons from 4 RCTs), no differences were found; the pooled odds ratio and 95% CI for mortality and nephrotoxicity was 0.89 (95% CI, 0.48-1.64) and 0.91 (95% CI, 0.56-1.47), respectively, comparing the continuous with the intermittent intravenous administration of antibiotics. Conclusion: Compared with the intermittent, the continuous intravenous administration of antibiotics is more efficient regarding clinical effectiveness. In an era of gradually increasing resistance rates among most pathogens, the potential advantages of the continuous intravenous administration of antibiotics on several clinical outcomes should be further investigated.

Penetration of moxifloxacin into rat mandibular bone and soft tissue Objectives: Odontogenic infections result in submucosal infiltrates and abscesses. They are able to penetrate into facial softtissues and bone. These mixed infections are mainly caused by both facultative and obligate anaerobes. Based on in vitro activity, the new 8-methoxyquinolone antibiotic moxifloxacin (MXF) seems to be suited for the antibiotic therapy of these odontogenic infections. The aim of this experimental study was to determine the levels of MXF in plasma, soft tissue (M. masseter) and the mandible. Methods: Forty-nine male fasted albino Wistar rats (aged 6 weeks) weighing 190-300 g were used throughout the study. A dose of 10 mg/kg MFX dissolved in water was administered orally per gavage. Blood samples of 7 animals (6 MFX, 1 control) were obtained 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 8 h and 24 h, respectively, after administration. The rats were killed with Ketalar (10 mg/kg i.m.) and tissue samples were collected. After homogenization (tissue samples) and protein precipitation all samples were analysed by HPLC/fluorescence detection assay. The pharmacokinetic evaluation was conducted based on noncompartmental analysis.

Results: MFX concentrations above lower limit of quantitation were measured from 0.5 h up to 8 h after administration. The exposure in terms of AUC (0 -1) amounted to 1821 lg h/L (plasma), 2463 lg h/kg (mandibles) and 5192 lg h/kg (M. masseter). The concentration-time curves of tissues were more plateau-shaped compared to plasma. Calculated AUC ratios tissue:plasma were M. masseter:plasma = 2.85 and mandibles:plasma = 1.35. Conclusion: Administration of antibiotics is considered as an important part of therapy during and/or after surgical procedures in the maxillofacial area. Because of its good penetration into bone and muscle tissues demonstrated in Wistar rats, MXF might be an option for clinical application. Based on the experimental data, controlled studies have to evaluate the clinical efficacy of MXF, also compared to other antibiotics.

Pharmacokinetic and pharmacodynamic aspects of high dose oral levofloxacin therapy for bone infections P. Viale, F. Pea, L. Scudeller, F. Pavan, M. Crapis, A. Londero, F. Cristini, M. Furlanut (Udine, I)

Objectives: To assess pharmacokinetic and pharmacodynamic aspects of high dose oral levofloxacin in the therapy of bone infection due to fully susceptible organisms.

Methods: All patients with microbiologically documented bone infection, treated at our Institution from 01/04 to 10/04 with oral levofloxacin, were included in the present analysis. Two regimens were routinely used according to age, body weight and renal function: 500 mg bid or 750 mg qd. According to different isolates other active antibiotics were administered. Patients underwent weekly clinical, biochemical and plasma therapeutic drug monitoring (TDM). Dosage adjustments were made according to plasma levels to maintain optimal exposure. Outcome was assessed at 8 weeks of treatment (16 weeks for discitis) and at least 1 month after the end of treatment.

Results: Fifteen patients were included. Mean age was 60 years (SD 17). 10 were males. Mean weight was 77 kg (SD 13). Median creatinine clearance was 91 ml/min (range 41-155). 7 pts had a prosthetic joint infection, 5 a post-traumatic ostheomyelitis (PTOM), 2 spondilodisicitis and 1 septic arthritis. Infections were monomicrobial in 13 cases (4 MSSA, 6 CoNS, 1 each E. faecalis, E. coli, K. ozaenae); polymicrobial in 2 (CoNS and P.aeruginosa, MSSA and MRSA). All but one isolates (MRSA) were levofloxacin-susceptible (available MIC for 9 isolates ranged 0.04-0.5 mcg/ml). Mean administered dosage was 8.9 mg/kg/daily (SD 2.2) . Average C min and C max during the overall treatment were 1.4 and 7.7 mcg/ml, respectively. Average C max /MIC ratio in the five patients with a punctual MIC was 133.21. Median treatment duration was 62 days, (range 17-135). At 1 month follow up after the end of treatment, 12 pts were deemed cured (clinically and by C Reactive Protein normalization). The 3 patients in whom levofloxacin failed 2 were prosthetic joint infections in which infected prosthesis was not removed, and 1 PTOM. No drug withdrawal was necessary due to adverse events, but 3/15 pts experienced mild arthralgies.

Conclusion: In our experience,TDM tailored high daily dose oral levofloxacin enabled adequate plasma levels and high cure rates, with no significant adverse events throughout long-term treatment. Plasma drug level monitoring is an exceedingly useful tool in the management of bone infections, suggesting dose reduction in pts at risk of adverse events and dose increase in pts suboptimally exposed.

Penetration of levofloxacin in sinusal mucosa of patients with chronic sinusitis following oral administration of a single 500 mg dose F. Pea, G. Marioni, F. Pavan, R. Bottin, A. Staffieri, M. Furlanut (Udine, Padua, I)

Objectives: To assess levofloxacin penetration in sinusal mucosa of patients with chronic sinusitis undergoing functional endoscopic sinusal surgery (FESS). Methods: Seven patients (mean ± SD; age, 45 ± 18 yrs; weight, 77 ± 13 kg; sex, 5/2 M/F; CLCr, 1.28 ± 0.32 mL/min/kg) were administered a standard 500 mg single oral dose of levofloxacin at 1 h (n = 4), 2 h (n = 1) or 3 h (n = 2) before surgical intervention. Intraoperative mucosal tissue and blood samples were collected simultaneously in each patient during FESS with the intent of assessing and comparing plasma and tissular concentration of levofloxacin. Levofloxacin concentrations in plasma and tissue homogenate were analysed by means of a validated HPLC method.

Results: Levofloxacin concentrations in plasma (mcg/mL) and tissue homogenate(mcg/g), respectively, after 1 h ranged 0.43-8.52 mcg/mL, and 0.78-9.87 mcg/g; after 2 h were 7.27 mcg/ mL and 8.91 mcg/g; and after 3 h ranged between 3.59 to 5.14 mcg/mL, and 6.46 to 9.34 mcg/g. Tissue-to-plasma ratio of levofloxacin at different sampling times ranged between 1.16 and 1.82. Conclusion: These preliminary findings suggest that single 500 mg oral dose of levofloxacin may ensure therapeutically effective concentrations in the sinusal mucosa of patients with chronic sinusitis. Of note, considering that fluoroquinolones exhibit concentration-dependent bactericidal activity and that optimal exposure may be achieved when peak-to-MIC ratio > 8-10 has been achieved, these data enable to conclude that a single 500 mg oral dose of levofloxacin may ensure effective exposure in sinusal mucosa against bacterial pathogens with an MIC < 0.8-1 mcg/ml, that is a value lower than the MIC90 of most pathogens usually responsible for bacterial sinusitis. Accordingly, these pharmacokinetic data are consistent with levofloxacin 500 mg once daily to be an effective antibiotic in the treatment of bacterial sinusitis.

A situation that mimics viral hepatitis: statin induced rhabdomyolysis fluvastatin 80 mg/day for hypercholesterolemia for a year which was changed to simvastatin 40 mg/day for the last two months by her doctor. The biochemical tests revealed that serum CPK was 119.300 U/L in 1/40 diluted serum sample, myoglobin was 4.000 ng/ml, viral hepatitis panel and Leptospira microagglutination test was negative. Her renal functions rapidly deteriorated. She was diagnosed as statin rhabdomyolysis and underwent to hemodialysis. The patient slowly improved and recovered with hemodialysis and discontinuation of statin drug. The incidence of rhabdomyolysis with statin monotherapy was reported as 0.04-0.2%. Our patient was taking high dose statin and was unaware of side effects. All patients taking statins should be informed about the side effects of these drugs.

The rationale for higher dose levofloxacin for the treatment of serious infections J. Kahn, N. Davis (Raritan, USA)

Background/Objective: Advances in medical care continue to be associated with improved survival rates and better patient quality of life. A consequence of these developments is an increased vulnerability to serious infection due to traditional pathogens (or commensals and relatively avirulent organisms) that may be resistant to multiple antimicrobial agents. There are few new antibacterial agents on the horizon. One strategy for dealing with this threat is to maximize the utility of each of our antibacterial drugs by dosing them more rationally. The purpose of this paper is to summarize the rationale for utilizing higher doses of levofloxacin in the management of serious infections. Methods: We have reviewed and will report on all available data from subjects exposed to daily levofloxacin doses of 750 mg or more. Results: After more than seven years of use in the US, levofloxacin has maintained its excellent susceptibility profile. Drug levels seen in infected patients are significantly higher than those achieved in healthy volunteers and enable coverage of organisms with higher MICs. Because of its concentrationdependent bactericidal activity, higher doses of levofloxacin increase the speed and thoroughness of bacterial eradication in vitro and can inhibit the emergence of fluoroquinolone resistance in studied organisms. Clinical trials with the 750 mg dose have been undertaken in complicated skin and skin structure infections, nosocomial pneumonia, and febrile neutropenia; results were at least equivalent to comparator agents. Use of this dose in community-acquired pneumonia and acute bacterial exacerbations of chronic bronchitis has allowed for more rapid resolution of symptoms and a shortening of the course of therapy. Dosing in healthy volunteers at does up to 1500 mg per day resulted in no significant alteration of the adverse event profile. Clinical trial data show that the safety of 750 mg daily is similar to the 250 and 500 mg forms.

Conclusion: Levofloxacin has documented efficacy and safety credentials at clinical doses of at least 750 mg once daily. At these higher doses, it is an attractive candidate for monotherapy of some serious infections or for combination therapy with such other agents as the carbapenems or 4th generation cephalosporins. Additional clinical trials using these approaches seem warranted.

In vitro activity of antimicrobial agents Introduction: Pseudomonas stem consists of resistible, aerobian, Gram-negative, non fermentation bacilli. They might cause various human infections and they are most frequent agents of intrahospital infections. Purpose: Investigation of Pseudomonas strains sensitivity to meropenem and imipenem. Material and methods: At the IGO CCS in Belgrade, from January 1st, 2004, till September 16th, 2004, a total of 131 strains of Pseudomonas from various patients' material were isolated. In 98 isolated strains, sensitivity to meropenem and imipenem was investigated by Gel Diffusion Technique, using commercial antimicrobial susceptibility test discs -Oxoid.

Results: Sensitivity to meropenem and imipenem was registered in 52/98 (53.06%), while 32/98 (32.65%) showed sensitivity to meropenem and resistance to imipenem. Another 10/98 (10.2%) of isolated strains were resistant to both antibiotics subject of this study. In 4 cases (4.08%), sensitivity to meropenem (S) was investigated, while sensitivity to imipenem was not tested. Out of 131 isolated strains, 79 were Pseudomonas aeroginosa, and in 61 strains, sensitivity to both meropenem and imipenem was tested. 36/61 (59.01%) showed sensitivity to both antibiotics. 16/61 (26.22%) were sensitive to meropenem and resistant either intermediary resistant to imipenem. 7/71 (11.47%) were resistant to both investigated antibiotics. 52/131

were Pseudomonas species, and in 37, sensitivity to meropenem and imipenem was investigated. 16/37 (43.24%) were sensitive to both antibiotics. 16/37 (43.24%) showed sensitivity to meropenem and resistance to imipenem. 3/37 (8.1%) isolated strains showed resistance to both investigated antibiotics. Conclusion: Isolated Pseudomonas strains demonstrate a high resistance to imipenem. In the investigation period, Pseudomonas strains resistant to meropenem were isolated for the first time ever. Considering that karbapenems are drugs of choice for the treatment of multiresistant strains, appearance of high resistance points to the necessity of their rational use.

The bacterial causes of nosocomial infections associated with short and long term-catheter the study. The most effective antibiotic against S.aureus was vancomycin (100%). For Gram-negative bacilli, the most effective antibiotics were amikacin (for E. coli), tobramycin (for P. mirabilis), cefotaxime (for K. pneumoniae) and ceftazidime (for P. vulgaris and E. cloacae). High level of antimicrobial resistance was noticed among P. aeruginosa, the most active antibiotic was amikacin. ESBL production was nearly the same in both NCL and community acquired infection, and it was highly prevalent among isolates of P. mirabilis (26%), followed by E. coli (8%). Other isolates showed no ESBL activity. Rough comparison of susceptibility results of E-test (ceftazidime strip) with that of disc diffusion and agar dilution methods in detection of the susceptibility test of the organisms to the antibiotic used. As regard the plasmid profile, it seemed that there is no particular association between the plasmid content and ESBL activity of the isolated nosocomial strains of both E. coli and P. mirabilis to explain interspecies relationship. Objectives: Bacteraemia represents a serious complication in hospitalized patients. Problems of therapy of bacteraemia increase with the emergence of antibiotic resistance. The aim of this study was to monitor the prevalence of pathogens and trends of resistance in bacteria isolated from blood. Methods: Six University Clinics and/or Regional Hospitals have participated in the study and a total of 1795 isolates were collected during 2002-2004. Microbiological diagnosis was performed according to standardized methods in participating laboratory. Antimicrobial resistance was estimated by the disc diffusion method by NCCLS.

Results: The most prevalent organisms were coagulase negative staphylococci (CONS) (28.7%), Staphylococcus aureus (14%), among Gram-negative bacteria Escherichia coli (13.4%), Klebsiella pneumoniae (9.3%) and Pseudomonas aeruginosa (6.4%) followed by enterococci, Enterobacter spp. and Acinetobacter spp. All CONS and S. aureus were susceptible to vancomycin, resistance to oxacillin was observed for 55-74% of CONS and only for 2-19% of S. aureus isolates. Enterococcus spp. isolates were fully susceptible to vancomycin and teicoplanin. Resistance to amoxicillin-clavulante increased from 9 to 35% among E. coli. Ciprofloxacin-resistance of K. pneumoniae isolates increased from 19 to 41%, despite this, ciprofloxacin together with carbapenems (100% susceptibility) was the most effective drug against K. pneumoniae. The most effective antibiotics against P. aeruginosa were meropenem, amikacin, piperacillin/tazobactam. Resistance to ceftazidime increased from 13 to 40% among P. aeruginosa. Imipenem retained 100% activity against Enterobacter spp. isolates, considerable was increasing of resistance to ciprofloxacin (from 15 to 58%). Resistance to meropenem, amikacin, ampicillin/sulbactam and cefoperazon/sulbactam in Acinetobacter spp. was relatively low.

Conclusions: Staphylococci, i.e. CONS and S. aureus have been identified as the most frequent causal agents of bacteraemia during all study period. The most significant rise in resistance was observed in ciprofloxacin against Enterobacter spp., E. coli and K. pneumoniae. Considerable is still good activity of carbapenems in Gram-negative and 100% efficiency of vanco-mycin and teicoplanin in Gram-positive bacteria. Surveillance of antibiotic resistance provides data about efficiency of antimicrobials in given locality and is important part of measures maintaining good antibiotic activity.

Comparative in vitro activity of ertapenem against ESBL-producing E. coli and K. pneumoniae isolated in Spain J. R. Hernandez, L. Romero, C. Velasco, L. Martinez-Martinez, A. Pascual (Seville, E)

Objectives: To study the in vitro activity of ertapenem against ESBL-producing E. coli and K. pneumoniae isolated in Spain.

Methods: E. coli (289) and K. pneumoniae (174) isolates from a Spanish multicentre study performed in 2000 (40 hospitals) and from the University Hospital Virgen Macarena in Seville (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) were included. ESBL production and antimicrobial susceptibility (betalactams, betalactams + betalactamase inhibitors, ciprofloxacin, aminoglycosides and co-trimoxazole) was determined by microdilution (NCCLS guidelines). Ertapenem activity was analysed according to clonal relationship (REP-PCR), ESBL-type (PCR and ESBL-gene sequencing) and acquisition of infection (community or nosocomial). Results: The in vitro activity of ertapenem was as follows: (Graphic 1)

All the isolates were susceptible to carbapenems. Resistance (resistant + intermediate isolates) of ESBL-producing E. coli and K. pneumoniae were: piperacillin-tazobactam 22% and 53%, ciprofloxacin 71% and 34%, co-trimoxazole 65% and 60%, amikacin 8% and 7%, gentamicin 36% and 48%, and tobramycin 44% and 63%, respectively. No significant differences were detected among the clones found. Ertapenem was equally active against all ESBL families (SHV, TEM and CTX-M) evaluated. Only four E. coli and one K. pneumoniae isolates had a MIC of ertapenem >0.5 lg/ml but still in the susceptibility range. Excluding these isolates, all coming from hospitalised patients, no difference in activity was observed according to the origin of infection.

Conclusions: Ertapenem showed an excellent in-vitro activity against ESBL-producing E. coli and K. pneumoniae. Ertapenem activity was similar to that of imipenem and meropenem but higher than that of other antimicrobials tested. No differences in activity were observed according to geographical origin, clonal relationship and type of ESBL.

Effect of beta-lactamase-inhibitor concentrations on in vitro test results with piperacillin/ tazobactam and piperacillin/sulbactam previously 8 mg/L. The present study was performed to assess the effect of this change on the MIC distributions of E. coli for Piperacillin/Sulbactam (P/S) as compared to Piperacillin/ Tazobactam (P/T) with Tazobactam tested at a concentration of 4 mg/L. Methods: The in-vitro activity of Piperacillin without and with Tazobactam or Sulbactam, respectively, against E. coli (n = 2856 in Leipzig, n = 420 in Berlin) was tested by means of microdilution MIC determinations as recommended by DIN 58940 using a fixed concentration of 4 mg/L of Tazobactam and 4 as well as 8 mg/L of Sulbactam.

Results: On the basis of MIC breakpoints according to DIN, the superiority of P/T over P/S is maximal in Piperacillin-resistant E. coli and minimal in Piperacillin-intermediate E. coli. The recently recommended reduction of the fixed Sulbactam concentration (from 8 to 4 mg/L) in susceptibility tests leads to reduced susceptibility in Piperacillin-intermediate E. coli, too.

The results are as follows: (table) Conclusion: The recently recommended 4 mg/L fixed concentration of Sulbactam in testing procedures shows that the in-vitro activity of P/T exceed those of P/S not only in Piperacillin-resistant but also in Piperacillin-intermediate E. coli. These results are credible because of the stronger inhibition of TEM-1 lactamases by Tazobactam in comparison to Sulbactam.

The inhibition of microbial adhesion in the presence of immune compounds on the human cell culture model Objectives: To comparatively evaluate the influence of the commercial preparations of normal human immunoglobulin (Russia), Intraglobin and Pentaglobin (Biotest, Germany) on the clinical strain S. aureus adhesion to the human embryonic skin fibroblasts in culture.

The clinical isolate of S. aureus with the highest adhesive capacity was selected in the model of it's co-incubation with the target cells in culture. The inoculums was prepared at a baseline concentration of 108 cfu/ml after 24 h grown on meatpeptone agar. The antiadhesive activity of the normal human immunoglobulin (NHI), Intraglobin and Pentaglobin at a baseline concentration of 20 lg/ml was assessed. The estimation of the influence of the agents under investigation on the bacterial adhesion process was performed on the above mentioned model of co-incubation of microorganism and the target-cells in culture as well as using 1 h pre-incubation with agents to be tested. Also the experiments were held in the presence Protein A from the staphylococcus cell wall as well as clostridial collagenasum and animal hyaluronidasum. The intensity of bacterial adhesion and antiadhesive properties of used compounds were expressed as following indices: Index of adhesion (IA) calculated as mean number of attached microbes per one eukaryotic cell; Percent of affected cells of the monolayer (AC%); Microbial contamination of the monolayer (MC) obtained as a product of IA•AC%; Percent of inhibition of bacterial adhesion (II%) calculated from the figures of IA with respect to the control. Results: All preparations revealed more or less significant inherent antiadhesive activity in vitro, which became apparent in the reduction of all indices characterizing colonization of target-cells.

Most effective proved to be the preparations with 1 hour preincubation with clinical isolate of S. aureus which protected fibroblasts against the bacterial adhesion in more than 92% in this case. Conclusion: The protective effect of NHI and Intraglobin disappeared with addition of Protein A, collagenasum or hyaluronidasum to nutrient medium but remained the same for Pentaglobin. According to its specific polymeric structure and prospective form as well as content of IgM Pentaglobin possesses much more antiadhesive activity in the presence of Protein A, collagenasum or hyaluronidasum. In the presence of Protein A and enzymes IgM makes the antiadhesive activity of Pentaglobin irreversible.

EDTA (ethylenediamine tetraacetic acid) enhances the activity of tetracycline in P. aeruginosa by increasing drug accumulation A. Sudano Roccaro, A.R. Blanco (Catania, I)

Objectives: In this work we investigated the potentiating effect of EDTA on tetracycline, versus resistant P. aeruginosa strains. Methods: We first assessed, through MIC determination, the effect of EDTA/tetracycline association on two tetracycline resistant P. aeruginosa strains. Next, through fluorescence microscopy, we evaluate the accumulation of tetracycline inside bacterial cells in presence or not of EDTA; moreover, preexposing cells to EDTA and successively (after washing them) to tetracycline, we evaluate if differences in tetracycline accumulation in presence of EDTA could be due only to its effect on the bacterial cell wall.

Results: Data show as EDTA, at sub-MIC concentrations is able to reduce the MIC for tetracycline. Fluorescence microscopy shows as cells, in presence of EDTA, accumulate much more tetracycline inside them respect cell directly exposed to tetracycline alone and also respect cells pre treated with EDTA and successively exposed to the drug. Conclusion: Thus we argue that EDTA is able to improve tetracycline MICs of resistant P. aeruginosa strains, not only permeabilizing the external cell wall, but increasing in other ways the bacterial tetracycline accumulation, perhaps interfering with the drug extrusion machinery. Abstracts discontinued. Routine susceptibility testing is performed in few and specialized laboratories only. Therefore data on activity of both substances are limited. The potency of resistance development against vancomycin and metronidazole is not known. Linezolid is a new antimicrobal agent active against grampositive bacteria. Methods: Selection of resistant mutants of the ATCC strain 43255 (MIC in mg/l for metronidazole, vancomycin, linezolid: 0.064, 0.5, 0.75) was performed on supplemented agar plates containing increasing amounts of the antimicrobials. Bacteria collected from five agar plates (after 48 h growth) were collected and centrifuged. The pellet was re-suspended and exposed to the next higher antimicrobial concentration on agar plates. Results: Repeating this procedure several times 7, 2 and 3 generations of bacteria for metronidazole, vancomycin and linezolid were generated, respectively. The bacteria were finally exposed to 5 mg/l metronidazole 2.0 mg/l vancomycin and 5 mg/l linezolid. Using Etest MHC values of 1.5 (23 · MHC), 2 (4 · MHC) and 2 (2.7 · MHC) mg/l could be confirmed. Conclusions: TIG is a potent compound with a wide spectrum of activity against Gram-positive and Gram-negative problematic bacteria, with the exception of P. aeruginosa. It could be an alternative to colimycin against multiresistant isolates causing infections in ICU patients.

The antibiotic resistance and metallo-b-lactamase production of carbapenem-resistant pseudomonas and acinetobacter Objectives: Pseudomonas aeruginosa infections are the major cause of morbidity and mortality in cystic fibrosis (CF) patients. Rates of multiresistance among Pseudomonas aeruginosa strains isolated from CF patients are substantially higher than among Pseudomonas aeruginosa strains from other settings. Mutator strains are characterized by increased mutation rates mainly due to defects in genes involved in the DNA mismatch repair system. In a previous study a link between high resistance rates in CF patients and the presence of a high portion of mutator Pseudomonas aeruginosa strains was found.The objective of our study was to investigate the proportion of mutators among multiresistant Pseudomonas aeruginosa from CF patients in Germany. Methods: Multiresistant Pseudomonas aeruginosa strains from CF patients were collected in a German hospital in 2004. Only one isolate per patient was permitted. Multiresistance was defined as reduced susceptibility (intermediate or resistant according to DIN guidelines) to at least four of the following class representatives: imipenem, ceftazidime, piperacillin, ciprofloxacin and gentamicin. The detection of mutators was performed by disk diffusion. Mutation frequencies were determined in triplicate on selective rifampicin agar plates. As previously defined, strains were designated hypermutable when the mutation frequency was at least 20-fold higher than that of the wildtype strain PA01.

Results: A total of 26 multiresistant Pseudomonas aeruginosa strains was collected. Of those 12 (46%) had increased mutation rates ranging from 5.85 · 10 )7 to 1.43 · 10 )5 . Eight mutator strains showed reduced susceptibility to all five and four mutator strains to four class representatives.

Conclusions: Comparable to a previous study, in Germany the rate of mutator strains is also extremely high among multiresistant Pseudomonas aeruginosa isolates from CF patients. Our data underlines the link between hypermutation and the evolution of antibiotic resistance in CF patients. Objectives: To investigate the frequency of resistance of P. aeruginosa to fluoroquinolones and to detect gyrA gene mutations in quinolone-resistant P. aeruginosa isolates obtained from discharge of sinuses, middle or external ears of patients.

The 126 isolated strains were tested for susceptibility to various antibiotics and those resistant to ofloxacin were tested for point mutations in the gyr A gene using single strand conformational polymorphism analysis ( SSCP, and by sequencing in 2 selected strains. Results: Twenty-eight (22.2%) out of the 126 isolates were resistant to ofloxacin. Point mutation in gyrA gene was not detected in any of the 28 quinolone resistant strains by SSCP. However, by direct sequencing of the PCR-amplified gyrA fragment from two clinical isolates, single mutation in which, threonine at position 83 was substituted by an isoleucine, was detected in both cases. Cells attached to surfaces in biofilms can be more tolerant to various anti-microbial agents than their planktonic counterparts. In biofilm environments the cells have the ability to differentiate and change phenotype leading to severe treatment problems in many cases of persistent or chronic infections. An approach addressing this issue identified specific resistant subpopulations in Pseudomonas aeruginosa biofilms and indicated that differentiation plays an important role in this context. Biofilms were established in flow-chambers, and their development and responses to addition of colistin (a polymyxin) were monitored by confocal scanning laser microscopy and time-lapse recording together with use of live/dead staining. Initial results indicate that modification of LPS is an important factor in relation to colistin tolerance development and may be the initiator of cell differentiation as pmr mutant biofilms of PAO1 result in sensitivity to colistin.Results indicated furthermore a regulatory connection between the composition of the cell envelope and cell motility (twitching, swarming) and in that way a correlation between motility and tolerance to anti-microbial agents. Tube biofilm experiments were set up for isolation of colistin treated and non treated biofilm material and further analysis of changes happening in the differentiating population by use of micro Array and LPS analysis.

Epidemiology of resistance, antibiotic usage and oxacillin 26 (20.2%); followed by lincosamides (lincomycin) 17 (13.2%), aminoglycosides 8 (6.3%). To notice, aminoglycosides were prescribed to 51 (17.0%) of all patients who received antibiotics. Body weight was determined in 6 (11.8%) and serum creatinine was measured in 37 (72.5%) of patients receiving aminoglycosides. Among prevailing combinations were gentamicin plus oxacillin 12 (14.6%), lincomycin and oxacillin 11 (13.4%), metronidazole and ciprofloxacin 5 (6.1%), and ampicillin plus gentamicin 5 (6.1%) of cases. In total, aminoglycosides for a combination therapy were administered in 43 (52.4%) of patients. Conclusion: Penicillins remain most often prescribed antibiotics for monotherapy and in combination with aminoglycosides. However surgeons often omit safety recommendations of administration of aminoglycosides. Further strategies to improve safety issues of antimicrobial therapy in surgery should be implemented.

Prolonged regional arterial antibiotics infusion in the management of severe acute pancreatitis I. Bihalskyy, S. Chooklin, A. Perejaslov, T. Ivankiv, O. Granat (Lviv, UKR)

Objectives: Development of the purulent-septic complications is the main cause of the death of patients with severe acute pancreatitis (SAP) after first week of the disease onset. The adequate antibiotics prophylactic is necessary part of the management of these patients. However, the disorders of microcirculation, which are typical for necrotizing pancreatitis, do not permit achieve the therapeutical concentration of antibiotics in inflamed pancreas. Due to that the continuous regional arterial infusion may have benefits in the management of patients with necrotizing pancreatitis. Methods: Ninety-four patients with SAP were treated. In 25 patients the antibiotics (ceftriaxone, gatifloxacin and imipinem) administrated arterially. These patients compiled the first group and the rest patients -the control group.

Results: The Balthazar, Ranson (more than 3) score was not different in both groups at admission. The rate of contamination of the necrotic foci was compared. During the first week the contamination of the necrosis was noted in 5 (7.2%) patients of the control group. During the second week the purulent complications of SAP were noted in 15 (21.7%) of the control group and one patients (4.0%) of the first group, which received ceftriaxone.

Conclusion: The continuous intraarterial infusion obviously decreases the risk of contamination of the necrotic foci. Applying gatifloxacin in the complex management of SAP has a benefit in prevention of purulent complications. Amoxicilin/clavulonic acid-86%, Gentamicin-86%, Cefalotine-83% and resistance for: Azlocilin-64%, Nitrofurantoin-54%, Neomicin-52% were found. Taking under consideration all micro-organismsÕ drug-sensitivity we observed that most useful in general practice was Ciprofloxacin-78% of sensitivity, Norfloxacin -78% and Amoxicilin/clavulonic acid-68%. Conclusions: In more than 50% of women under GP care the UTI etiological factor was E. coli. Fluoroquinolones and amoxicilin/clavulonic acid should be preferable drugs to use in GP, considering their wide-spectum activity. Considering costs, drug-sensitivity examination should be limited to antibiotics most frequently used in GP.

Extended spectrum b-lactamase producing Escherichia coli in a Kuwait hospital Objectives: Since the 1990s the CTX-M type b-lactamases have constituted one of the most rapidly spreading extended-spectrum b-lactamase (ESBL) families. They have been detected in many countries of the world. The aim of this study was to genetically characterise the phenotypically ceftazidime susceptible and cefotaxime resistant ESBL-producing Klebsiella pneumoniae isolates. Methods: Species identification was performed with BBL Crystal E/NF, and ESBL expression was confirmed with E test ESBL. CTX-M genes were amplified with PCR using consensus CTX-M primers. The conjugation experiments were performed using Escherichia coli J53-2 (rifampin resistant). The CTX-M genes in the transconjugants was detected with PCR. The amplifield products were analysed with RFLP, assigned to distinct groups on the basis of RFLP profiles and further analysed using specific primers. The amplified products were sequenced. The isolates were also investigated by phagetyping, genomic fingerprinting by ERIC-PCR and PFGE and plasmid profile analysis. 

Setting up a surveillance system of antimicrobial resistance and use in a low-resource setting: West Delhi, India Objectives: To set up a surveillance system for antimicrobial resistance and use of antimicrobials in the community in a low resource setting using E. coli as an indictor organism. Methods: The study area was ÔBlocksÕ in a 10 Km. radius of Sir Ganga Ram Hospital, New Delhi, India. Microbiology Surveillance: MSU's were collected from all cases not taking antimicrobials and without suspected complicated UTI attending the OPD from designated region. Once E. coli (pathogenic as well as contaminants) were isolated and identified using conventional biochemical reactions its susceptibility was determined using Kirby Baur disc diffusion method on Muller Hinton agar. Antimicrobial use surveillance: Monthly purchase of all antimicrobial drugs from 30 private retail pharmacies was collected and converted to DDD. The denominator for the drug use was the population of the administrative areas in which these pharmacies are based. In addition, exit interviews (prescription auditing) were done at each pharmacy to have information on 15-25 consecutive prescriptions per month having any antimicrobial agent. Data on use is presented as DDD's per total number of patients/prescriptions. Results: During the study period (11/03-9/04) 3590 MSU samples were collected and 377 E. coli were isolated. 85% of the strains were resistant to ampicillin, and more than 50% were resistant to quinolones (60%), tetracycline, third generation cephalosporins, cotrimoxazole and ampicillin + sulbactam. Purchase data and exit interview data was analysed for 9 months and the results showed that fluoroquinolones are the most frequently sold antimicrobials. Exit interview data revealed that DDD/1000 prescriptions/patients/month for fluoroquinolones; penicillins, cephalosporins and macrolides are 2972,1296, 1034,884 respectively. Conclusions: The extensive usage of quinolones and penicillins are reflected in the antimicrobial resistance. A decrease in resistance to cephalexin from 85% to 41% could be noticed during the study period. The data collected on antimicrobial drug use and antimicrobial resistance in E. coli isolated from antenatal cases seems suitable for collection of information on the resistance and use in the community, valuable for planning targeted interventions. This is an on going project for one year. Funding agency: WHO, Geneva.

Resistance to telithromycin in Streptococcus pneumoniae is rare and associated with reductions in bacterial fitness D.J. Farrell, J.G. Hurdle, I. Chopra (London, Leeds, UK)

Objectives: PROTEKT -a global, longitudinal study of the antibiotic susceptibility of bacterial respiratory tract pathogenshas now completed its fourth year. The objectives of this study were to determine the global rates of bacterial resistance to telithromycin, the first ketolide antibiotic, and to evaluate the fitness of two telithromycin-resistant Streptococcus pneumoniae isolates relative to their putative isogenic telithromycin-susceptible counterparts. Methods: Minimum inhibitory concentrations (MICs) for telithromycin were determined by the NCCLS broth microdilution method and interpreted using NCCLS breakpoints. The isogenic relationships between telithromycin-susceptible and -resistant isolates (two independent sets) were determined by serotyping and multi-locus sequence typing. The relative competitive fitness (W) of telithromycin-resistant strains was evaluated by established procedures involving mixed culture competition between isogenic pairs of telithromycin-resistant and -susceptible strains.

Results: Of the 20 750 S. pneumoniae isolates collected between 1999 and 2003 in the PROTEKT study, 0.1% exhibited low-level resistance to telithromycin (MIC ‡ 4 mg/L), with the highest MIC being 8 mg/L. This low rate of telithromycin resistance is observed across the globe, ranging from 0% in Australasia, the Middle East and North America to 0.22% in Central Europe. The development of resistance to telithromycin was accompanied by moderate reductions in W -32% in one strain and 25% in the other. Conclusions: Clinical resistance to telithromycin among S. pneumoniae is rare and low level, with a global prevalence of around 0.1%. In the two sets of isogenic strains examined, resistance to telithromycin was associated with moderate fitness cost (25-32%). These data suggest that the potential for S. pneumoniae to develop clinically relevant resistance to telithromycin may be limited. However, further studies are needed to examine the mechanisms of telithromycin resistance and the potential for genetic adaptation, which might reinstate bacterial fitness in telithromycin-resistant strains. Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 function and for a shorter duration of therapy to improve bactericidal killing and post-antibiotic effect whilst reducing the risk of toxicity associated with prolonged treatment. We performed a prospective audit of gentamicin usage in our institution over a one month period. Objective: Our aim was to assess the sensitivity to antibiotics of the most commonly isolated pathogens in blood cultures from patients admitted in a tertiary care hospital. Materials and methods: We analysed the results of 858 positive blood cultures, during last year from patients admitted in Ahepa University hospital, focusing on their susceptibility to antibiotics. Identification of bacterial isolates and their antibiotic susceptibilities were performed by Vitek 2 analyzer of Bio-Merieux.

Results: The most frequent isolate was coagulase-negative staphylococci (CNS) (29%) followed by Staphylococcus aureus (16,9%), Acinetobacter calcoaceticus (12,8%), Pseudomonas aeruginosa (9,6%), E. coli (9,4%), Klebsiella pneumoniae (5,9%), and Proteus spp (3,5%) . Klebsiella spp and Proteus spp. isolates were not significantly resistant to the antibiotics used. E. coli isolates were resistant as followed: 64% to ampicilline, 36 % to trimethoprime /sulfamethoxazole, 28% to amoxicillin/clavulanic acid (A/CA), 7% to cefuroxime, 19% to nitrofurantoin and 6% to ciprofloxacin (CIP). There was no resistance to ceftazidime (CFZ) and imipenem (IMI). Among Pseudomonas aeruginosa isolates 26% were resistant to CFZ, 28% to CIP, 22 % to IMI, 24% to tobramucin (TO) and there was no resistance to piperacillin/tazobactam (P/T). In terms of sites of isolation Acinetobacter spp were mainly isolated from surgical wards while Staphylococcus epidermidis spp from other clinics. 36% of Acinetobacter spp isolates examined showed resistance to ampicillin/sulbactame, 77% to IMI , 79 % to meropenem, 72% to P/T and 97% to CIP. 89% of CONS strains and 67% of S. aureus isolates were resistant to oxacillin. As far as glycopeptide resistance is concerned 88% of CONS isolates and 100% of S. aureus were sensitive to teicoplanin, while no VRSA or VRSE strains were isolated. Conclusions: One third of the S. aureus isolated in our hospital were methicillin resistant (33% MRSA). Compared with previous results there has been observed a significant increase in resistance rates of pseudomonas aeruginosa to all antibiotics except ceftazidime, as well as in Acinetobacter spp to piperacillin/ tazobactam.

Resistance to antibiotics and heavy metal in different species of Aeromonas collected from the Loa River in Chile 

What factor will influence fever in communityacquired pneumonia?

D. Genné, R. Sommer, R. Rakotoarimanana, D. Lew (La Chauxde-Fonds, CH)

Objectives: Although fever (> 38°C) accounts for one of the major clinical signs of community-acquired pneumonia (CAP), it is not well studied in that disease. Methods: We conducted a prospective observational study involving 228 adults patients hospitalized for CAP (diagnosed on IDSA guidelines) and measured their axillary body temperature 3 times a day until discharge. The causal pathogen, the empirical antibiotic choice and other factors that could influence the follow-up of fever were investigated. Results: 162 (71%) patients requiring a hospitalisation for a CAP presented with fever. Although the level of fever decreased with age, it was not statistically significant. The mean duration of fever was 3 days (1-28). Except the presence, in one instance, of influenza virus which shortened the duration of fever, there were no differences related to the causal agent. A multivariate analysis showed that the presence of empyema and the empirical use of the quinolone trovafloxacin prolonged the duration of fever of 3 days (CI 95% 1.5-4.7, p < 0.001) and 0.7 days (CI 95% 0.09-1.30, p = 0.024) respectively.

Conclusions: Fever was absent in a significant proportion of patients (29%) hospitalised for a CAP. It should last for about 3 days for appropriately treated patients. For those with a febrile period exceeding 5 days, the presence of empyema should actively be searched.

Peripheral T-cell lymphoma masquerading as cellulitis I. Bliziotis, G. Peppas, P. Rafailidis, P. Vergidis, A. Kapaskelis, P. Papastamataki, M. Falagas (Athens, GR)

Objectives: Primary cutaneous non-Hodgkin lymphomas (NHLs) encompass many subtypes of lymphoma, namely mycosis fungoides, lymphoblastic lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, anaplastic large cell lymphoma and peripheral T cell lymphoma not otherwise specified. However, there are no reports of cellulitis due to lymphoma. Methods: Description of our experience with a patient with peripheral T-cell lymphoma masquerading as cellulitis.

Results: A 28-year-old Greek-American male presented with extensive cellulitis of the left sub-axillary area. The patient had been evaluated in two hospitals abroad where he was advised to receive antibiotics, which did not lead to any appreciable improvement. A supraclavicular mass, thought clinically to be an enlarged lymph node, was removed at the second hospital. The biopsy showed inflammatory changes without any evidence for malignancy. Biopsy of a mass from the left axillary fossa, removed at our hospital, showed conclusive evidence for peripheral T-cell lymphoma.

Conclusion: A significant delay in the correct diagnosis was the result of a rare manifestation of lymphoma and limitations of the diagnostic work up, including biopsy of removed tissue. Clinicians should be aware that lymphoma may present with typical cellulitis-like lesions. Results: We report our experience with the management of a 44-year-old man with manifestations of Kyrle's disease. The patient presented with skin lesions of two different types on his face and upper and lower extremities. The old lesions were large hyperkeratotic plaques with burrows without signs of inflammation. The newer lesions were also hyperkeratotic plaques but they clearly had signs of inflammation. The patient was successfully managed with combined surgical and medical management. The hyperkeratotic old and large skin lesions, which did not have manifestations of ongoing inflammation, were surgically removed. In addition, antimicrobial therapy with oral clindamycin was administered for one month, which led to disappearance of the small newer lesions, which had findings of ongoing inflammation and were clearly part of Kyrle's disease. No recurrence was observed during 8 months of follow up. Conclusion: Although clindamycin may have some anti-inflammatory effects unrelated to its antimicrobial action, the observed control of the inflammation and the gradual regression of the newer lesions with clindamycin, suggest that microorganisms (probably anaerobic bacteria) play a role in the pathogenesis of the progression of Kyrle's disease at least in the initial stages of the skin lesions.

A prospective study of neck mass excluding thyroid nodules in one hospital

Introduction: A mass in the neck is a common clinical finding that presents in patients of all age groups. The differential diagnosis may be extremely broad, and although most masses are due to benign processes, malignant disease must not be overlooked. Therefore, it is important for physicians to develop a systemic approach for developing a working diagnosis and management plan for the patient. Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 Results: Thirty-two patients did not accept more evaluation, the findings of 123 patient left include: 47/3% men, 52/7% women, 43/6% single, 56/4% married; 8% less than 10-yearsold, 28% 10-19-years-old, 22% 20-29-years-old, 7/4% 30-39-years-old, 10/2% 40-49-years-old, 10/2% 50-59-years-old, 6/6% more than 60-years-old; time of neck mass in 24/1% less than one month, 29/9% 2-3 months, 21/8% 4-6 months, 10/3% 7-12 months, 13/8% more than 12 months; mass position 42/8% on left side of neck, 29/7% on right side of neck, 24/1% on both sides, 2/8% on midline, 0/7% on occiput; number of mass 59/4% one mass, 6/3% 2 masses, 34/3% 3 or more than 3 masses; also size of masses was evaluated. 30% have signs and symptom, the more common signs are lymphadenopathy on the axilar and hepatomegaly, 13/6% have past history. Objectives: The purpose of this study was to evaluate the effect of oral estroprogestinic hormone replacement therapy (HRT) on the bacterial vaginosis (BV) prevalence in postmenopausal women. Methods: One hundred thirty eight women, aged 45-69 years were enrolled in this study. All of them had a natural termination of menstruation in at least 12 months prior the study. Fifty eight (group A) were currently received oral HRT (tibolone), whereas the other 80 (group B) had not received HRT, orally or transdermally at least in the past six months. All vaginal speciments were collected from the posterior fornix or lateral vaginal wall. Diagnosis of BV mainstayed on the Nugent score method (a score of 7-10 was considered indicative of BV) as well as the presence of Ôclue cellsÕ on Gram stained vaginal smears.

The mean age ± SD of women in group A and the average number of postmenopausal years was 56.4 ± 3.9 years (range, 51-63 years) and 6.8 ± 3.1 (range, 4-13 years). While in group B was 55 ± 5.2 (range, 45-69 years) and 6 ± 3.2 (range, 1-18 years) respectively. Prevalence of BV in group A was slightly lower (12.5 %) than among women in group B (13.3 %), but the difference was not statistically significant (P > 0.05). Absence of lactobacilli from vaginal smears without any symptoms of vulvovaginitis was found in 36.6% of women in group B and 11% of women in group A. High number of lactobacillus colonization was observed in only 10% of women who did not received HRT.

Conclusions: Treatment of postmenopausal women with tibolone has no statistically significant effect on Ôfull BVÕ prevalence. However, it did seem to affect lactobacillus colonization of the vagina, altering the flora to premenopausal profile.

Bacterial pathogens associated with acute diarrhoea on the island of Crete, Greece, and their resistance to antibiotics Objective: To present a case of WellsÕ Syndrome due to tick bite.

Case report: A case of WellsÕ syndrome possibly due to tick bite is presented. Eosinophilic cellulitis (WellsÕ Syndrome) is characterized by skin eruptions with edema, flame figures and marked infiltrate of eosinophils in the dermis. Hypersensitivity to arthropod bites, cutaneous viral infections (HSV-2), cutaneous parasitic infestations (toxocariasis), ascariasis and drug hypersensitivity has been reported as etiologic factors. Sixtyyear-old woman was referred to our clinic with the suspect of erythema chronicum migrans and erythema annulare centrifugium. The patient complained from widespread reddish elevations since 5 months after tick bite. Lesions had first appeared in scalp and face, then had spread to the body. She was hospitalized in the local hospital; skin biopsy was consistent with nonspecific chronic dermatitis and anti-Borrelia IgM and IgG were positive. Terbinafine (systemic and local), systemic tetracycline, amoxicilline clavulanate was administered but no response was observed. The patient was hospitalized in our clinic with the initial diagnosis of eosinophilic cellulitis, erythema chronicum migrans and erythema annulare centrifugium. Dermatologic examination revealed widespread elevated erythematous papules and plaques of 1-10 cm diameter formed by coalescing papules, with central sparing.

Histopathological examination of punch skin biopsies showed eosinophilic infiltration in the dermis, but no flame figures were observed. Borrelia burgdorferi IgG was positive but IgM was negative. Marked eosinophilia (11%) was noted in the peripheral blood sample, total IgE was elevated (275 mg/dl) other biochemical parameters were normal. No other signs of systemic Borreliosis was found. The patient was treated with dapsone (150 mg/day) and methylprednisolone (80 mg/day) with the diagnosis of eosinophilic cellulitis. Lesions had tended to resolve by the 2nd month of the therapy and complete resolution without sequele was observed on the 6th month.

Conclusions: Tick bite has been reported as a causal factor for Wells' syndrome. Moreover, arthropod born infectious agents like Borrelia burgdorferi may also be a triggering factor. Dapsone combined with corticosteroids may be an effective treatment in this situation. The role of antibiotherapy in the acute phase of the infection is controversial.

Rabdomyolysis complicating Shigella flexneri acute gastroenteritis C. Papaefstathiou, A. Diakakis, K. Mitsouras, M. Marioli, G. Kouppari (Athens, GR)

Objectives: To report a case of acute rabdomyolysis associated with Shigella flexneri acute gastroenteritis.

Methods: Stool specimens cultured on the appropriate media for isolation of the enteropathogenic bacteria and direct smears were examined. The enteropathogenic bacterium was identified by standard methods, API 32E system (bioMerieux) and serotyping with antiserum reagents. Myoglobin was detected by biochemical method. Susceptibility testing was carried out by disc diffusion method. Case report: A male patient aged 71 years was admitted to the hospital because of acute gastroenteritis with watery, bloody stools associated with abdominal pain and myalgias. He was suffering from coronary artery disease and diabetes. The patient was pyrexial (380°C), dehydrated, hypotensive (blood pressure: 90/70 mmHg) and had generalized weakness. Abundant leukocytes and erythrocytes were seen on direct smears of stools. Admission laboratory data included WBC count 18.250/ll (neutrophils 84.6%), Ht 45.1%, Hb 15.9 g/dl, urea 64 mg/dl, creatinine 1.5 mg/dl, glucose 192 mg/dl, Ê 3.6 mEq/l, Na 134 mEq/l, CPK 11840 IU/L (normal value < 170 IU/L), CK-MB 124 IU/L (normal value < 6%), LDH 543 IU/L, SGOT 265 IU/L, SGPT 81 IU/L, ã-GT 21 IU/L, albumin 3.2 g/dl and myoglobinuria. From the clinical and laboratory findings rabdomyolysis was diagnosed. The patient was treated with parenteral hydration and ciprofloxacin 400 mg tid iv. From the stool culture S. flexneri isolated. The strain was resistant to trimethoprime/sulphamethoxazole, ampicillin, amoxycillin/ clavulanic acid, tetracycline, chloramphenicol and susceptible to ciprofloxacin, cefotaxime, piperacillin. His condition showed gradual improvement. At the next days he was presented gradual improvement. CPK on the 2nd and 3rd hospital day was 4700 IU/L 900 IU/L respectively and had returned to within normal by the sixth hospital day. The patient was discharged on day 7. Conclusions: Rabdomyolysis complicating S. flexneri gastroenteritis is very rare. The present case is only the second found in the English literature. The possibility of rabdomyolysis must be considered in all cases of severe gastroenteritis so that prevention and timely treatment of complications are made possible.

Corynebacterium urealyticum encrusted cystitis: a new case and review of literature Encrusted alkaline cystitis (EC) is an unusual process, almost forgotten by urologists. It tends to appear complicating an underlying cystopathy. EC consists of a vesical mucosal inflammation with encrustation of amonium magnesium phosphates in the urothelium. Their genesis requires pre-existing mucosal damage, urinary infection by urolithic microorganisms and alkaline urine. A 50-years-old man was victim of a traffic accident with multiple injuries. He was admitted in our ICU hospital and urinary catheterisation was perfomed for 20 days. Because of hematuriae and abdominal pain urine samples were collected. Corynebacterium urealyticum grown in the urine, and some foci of malakoplakia were found in the area of encrustation and endoscopically excised. Antibiotic treatment with teicoplanin and ciprofloxacin was fulfilled during 60 days. After 30 days of treatment, endoscopical resection of bladder wall was achieved for bacteriological data. Few cases of encrusted cystitis were reported this past years. We propose literature review and therapeutic options in this communication. Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 risk than in high risk Fine groups. (4) However, S. pneumoniae was significantly more common as severity increased. (5) H. influenzae did not show clustering in any group. (6) M. pneumoniae was more common in less severe cases. Objectives: Group B streptococci (GBS) are causes of lifethreatening infections in neonates, infants and pregnants ,but they are possible causes of invasive infections especially in immunocompromised adults. GBS arthritis has been rarely described in immunocompetent adults, usually associated with other risk factors as neoplasms, cirrhosis, diabetes. We present a GBS arthritis in an immunocompetent male patient. Susceptibility test was performed by disc diffusion method (according to NCCLS).The strain was resistant to aminoglycosides. Antibiotic therapy was changed to intravenous penicillin. Whole body bone scan, with Tc99m-MDP revealed arthritis of the right acetabulum. 8 days later, the patient presented increased mobility of the right hip and he finally recovered without sequelae.

Conclusions: This case report tries to increase the awareness of clinicians to GBS as a potential cause of septic arthritis. Although GBS disease is frequent in adults with aspleniaorganic or functional-, chronic liver disease, diabetes and congestive heart failure, and although urinary tract infection, pneumonia and soft tissue infections are the commonest presentations, GBS has not to be underestimated in the differential diagnosis of septic arthritis even in patients without predisposing factors.

Severe complications in patients with Brucella melitensis infection Brucellosis can present with a variety of symptoms and can affect virtually every organ and tissue of the body. We report here complications in 15(17%) out of 88 patients with Brucellosis that were diagnosed and treated at our Hospital in a period of 7 years. These complications were of clinical significance because they lead to diagnostic and therapeutic dilemmas.

Complications that lead to diagnostic dilemmas because of the broad list of differential diagnosis included skin rash in 3 patients, orchitis/epipidymitis in 3 patients, severe pancytopenia in 2 patients and single lineage cytpenias in another 3, and finally stiff neck with CSF pleocytosis in one patient.Therapeutic dilemmas emerged in 3 patients who developed arthritis (spondylitis, sacro-ileiitis and spondylitis, and right ankle arthritis, respectively) while they were receiving treatment for Brucellosis. Finally, it was only after a detailed diagnostic work up that a patient who presented with recurrent fever and arthritis while on treatment for Brucellosis for 8 weeks was diagnosed with endocarditis of an artificial aortic valve. Successful management required prolonged treatment with a combination of antibiotics. Conclusion: The diagnosis and treatment of Brucellosis are usually simple. However, the successful management of its complications that can be severe, may require detailed diagnostic work up and prolonged treatment. Methods and materials: We analysed records of patients clinical materials from recent 2 years. We present 4 posttraumatic patients with severe GAS infections of soft tissues with cellulitis and STSS. All patients developed infectious complications following injuries. All cases required quick and aggressive surgical procedures-large incisions and removal of necrotic tissues; one of the patients needed multiple surgical interventions. Three patients improved in spite of severe course of infection; one patient died. Two patients suffered from head injury, two patients had injuries to the extremities complicated by extended tissue necrosis. Amputations of the limbs were not necessary. The course of infections was rapidly progressive with fever, extensive soft tissue necrosis and marked systemic toxicity. All cases required broad spectrum antimicrobial therapy-patients were treated with lincosamids, mainly with clindamycin given intravenously in large doses from 1800 to 3600 mg/day. In all patients microbiological procedures were performed: cultures of the wound and blood were obtained and sent for routine and anareobic cultures and for direct microscopic examination. Suitable diagnostic materials were: exsudate from the wound and fragments of necrotic tissues.

Conclusion: GAS infections are severe and life threatening. According to our findings clindamycin in high doses improves the prognosis and should be the drug of choice in Streptococcal cellulitis and STSS.

Demographic and microbiologic features of urinary tract infections in young and adult patients Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 (10% vs 0.8%, p < 0.001 ). E. coli showed similar resistance rate in males and females to ampicillin, cotrimoxazole, nitrofurantoin and ciprofloxacin. The isolation rate of E. coli was lower (54.4% vs 64.2% , p < NS ). in women treated with antimicrobials during the 2 months prior their enrolment than in those with no previous treatment. The resistance rate of E. coli to the antimicrobials in previously treated and non treated women was the following respectively : cotrimoxazole 22.6% vs 8.3%, p = 0.07; ampicillin 41.9% vs 22.6%, p NS; ciprofloxacin 9.7% vs 0.4%, p NS ; nitrofurantoin 9.7% vs 8.8%, p NS).

Conclusions: In young and adult pts (16-50 years of age), E. coli is the causative agent of UTI more frequently in females than in males. In men, other microorganisms (expecially Pseudomonas ) play a significant role. In females, a previous antimicrobial treatment increases the risk of E. coli resistance to ampicillin and cotrimozazole.

Complications of acute brucellosis among adults in Diyarbakir, Turkey Objectives: Serious respiratory infections, due to P. aeruginosa and B. cepacia consist a major problem in the life of Cystic Fibrosis (CF) patients. In Greece, the B. cepacia isolation in CF is considered rather rare. The aim of our study was to investigate the presence of B. cepacia in respiratory infections of CF patients of ÔSotiriaÕ Chest Diseases Hospital of Athens.

Methods: The selective medium B. cepacia agar (Oxoid) was used, in addition to conventional media, for quantitatively culturing sputum samples of CF patients, admitted to our Hospital, for the period 2003-2004. Identification methods included API 20E, API NE, and Vitek 2 (Biomerieux). All Gram (-), oxidase (+) isolates were also molecularly identified initially by multiplex PCR, in order to identify two bacterial species P. aeruginosa and P. cepacia. The multiplex PCR included primers specific for the 520 bp fragment of the ribosomal 16S gene, identifying the five main gemovars of B. cepacia. Included in the multiplex PCR was a universal bacterial primer pair targeting the 16S rRNA to act as internal control (233 bp). Samples giving results for the 520 and/or 463 bp fragments were further tested for the identification of the bacterial strain with specific PCR. The recA gene PCR is a useful assay, which discriminates between the B. cepacia complex and B. cepacia like microorganisms.

Results: None of the isolates was identified as belonging to B. cepacia complex, either by selective medium or PCR. A high level of correspondence was noted between selective B. cepacia medium and molecular identification of isolates.

Conclusions: There is evidence that B. cepacia participation in CF respiratory infections is rather unusual in Greece.

Influence of proper antimicrobial prescribing on outcome of acute sinusitis

Objective: The aim of the study was to evaluate correlation between antimicrobial (AM) prescribing for outpatient adults with acute sinusitis (AS) and outcome of the disease. Methods: Case histories of outpatients with the diagnosis of AS were consecutively selected in outpatients' departments in 8 regions of Russia for retrospective analysis. AM were classified using ATC-codes. All collected data were analysed with specially designed software ÔPharmatherapeutical Data AnalysisÕ (IAC, Smolensk, Russia). Outcomes were classified as ÔsuccessÕ (cure or improvement) and ÔfailureÕ (hospitalization or prescription of an additional AM). Patient's age and frequency of sinus puncture (SP) as factors potentially influencing outcome of AS were also analysed. Ages were compared with Wilkokson-Mann-Whitney test. Outcomes and frequency of SP for the treatment with different AM were compared using weighted pairwise Fisher criterion.

Results: A total of 1529 case histories of outpatients aged from 16 to 81 (539 males, 990 females, average age 37.1 ± 13.2 years) were analysed. 10 most popular AM (82.7% from all AM) used for initial monotherapy including ampicillin (AMP), amoxicillin (AMX), amoxicillin/clavulanate (AMC), erythromycin (ERY), midecamycin (MID), gentamicin (GEN), lincomycin (LIN), doxycycline (DOX), ciprofloxacin (CIP), co-trimoxazole (CTX), were prescribed to 1029 (67.3%) patients. All groups were similar according to age. AMX group was associated with significant less frequency of SP than LIN, CIP, GEN (p < 0.001) and MID, AMC (p < 0.05) groups. Initial prescription of CIP or LIN was found to have higher rates of SP compared to AMP, ERY and DOX (p < 0.05). Rates of treatment success and failure are presented in the table. Treatment with AMX was statistically more successful than with other AM (p < 0.05) except AMC and MID. There was no significant difference in outcomes between other groups except MID, which had better outcome than CIP and DOX (p < 0.05).

Conclusion: proper AM prescribing (AMX) for initial treatment of AS led to better outcome. AMX start-up therapy was associated with less frequent SP. Amoxicillin + gentamicin (14.7%), ampicillin + gentamicin (9.2%), ciprofloxacin + gentamicin (5.5%) were the most frequently prescribed combinations in 2003 vs. ampicillin + co-trimoxazole (26.1%), gentamicin + co-trimoxazole (9.2%), ampicillin + gentamicin (6.7%) in 1998. In 72.5% of cases AM were administered orally (vs. 71.6% in 1998), in 24.7%intramuscularly (vs. 28% in 1998), 0.5% -intravenously, 2.3% -route was not indicated. Conclusion: Numerous AM were used for CAP treatment in 2003 as well as in 1998, but prescribing habits have improved. An increased rate of amoxicillin and amoxicillin/clavulanic acid and a decreased gentamicin and co-trimoxazole usage were observed. The very low rate of ÔnewÕ macrolide usage and an unacceptably large number of ciprofloxacin and cefazolin prescriptions should be noted. Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 events of clinical significance occurred. On day 84 of his hospitalization, the patient developed pneumonia due to Staphylococcus aureus and Pseudomonas aeruginosa strains. He was managed with intravenous antimicrobial agents including vancomycin, gentamicin, and piperacillin/tazobactam, which led to clinical improvement. However, on day 96, the abovementioned isolates grew again from cultures of bronchial secretions specimens. Gentamicin was discontinued, and intravenous ciprofloxacin and rifampin were started. On day 117, the patient developed an extensive maculopapular rash on the trunk and the extremities. In addition, ocular and perioral swelling was observed. All antimicrobial agents were discontinued. On day 119, he developed bacteraemia due to Klebsiella pneumoniae strain. Intravenous gentamicin was started but it was discontinued one day later due to the flare up of the rash. Finally, on day 127, a blood specimen culture grew a MDR Acinetobacter baumannii strain, sensitive only to colistin. A continuous intravenous infusion regimen with colistin 2,000,000 units per 24 hours was initiated. The clinical condition of the patient gradually improved, and the rash gradually diminished. Conclusion: Continuous intravenous administration of colistin may be associated with fewer hypersensitivity (adverse) reactions and may be as effective as the traditional intermittent way of infusion.

Colonisation and antimicrobial resistance in tracheal aspirates of hospitalised patients in intensive care units of a tertiary medical care centre in Kosova Results: Over the period of the study 801 sets out of the total 1431 blood cultures were positive for microbial growth, which represented 648 cases of neonatal bacteraemia from a total of 1092 cases. From the total number of isolates the members of enterobacteriaceae Serratia, Klesiella and Enterobacter spp were the most common cause of bacteraemia. The coagulase negative and positive Staphylococci were also frequently isolated. A changing pattern of causative pathogen was observed during this study between members of the three leading genera of Enterobacteriaceae. Antibiotic susceptibility testing showed a high level of resistance among the most common pathogens. Resistace to Aztreonam, Imipenem, Ciprofloxacin and piperacillin/tazobactam was less frequently encountered. Staphylococcus resistance to anti-staphylococcal antibiotic and due to hyperproduction of penicillinase enzyme was also high and all isolates were remained sensitive to vancomycin. Conclusion: Gram-negative bacteria especially members of the enterobacteriaceae are important causes of bacteraemia in neonatal intensive care units. Although most isolates remain sensitive to the new antibiotics, emergence of resistant strains can not be excluded in the future. For that reason new strategies and continuous surveillance are required to monitor the changing epidemiology of pathogens, antibiotic susceptibilities and antibiotic use needed to overcome the increasing incidence of resistance to conventional drugs.

Phagocytic activity of human peripheral blood polymorphnuclear leucocytes to methicillinsensitive and resistant coagulase-negative Staphylococci

Objectives: Two types of white blood cells, the polymorphnuclears (PMN) and monocytes are important in resistance to infections. Phagocytes play a significant part as the first line of defence against bacteria, viruses and fungi in the human host. Objectives: The aim of this study was to assess the frequency of colonization of nasopharynx by Gram-negative rods in patients with lung cancer undergoing thoracic surgery, who routinely receive antimicrobial prophylaxis (piperacillin, cefuroxime alone or in combination with amikacin). Antimicrobial agent susceptibility of isolated strains was also defined. Methods: Sixty-three patients with resectable lung cancer were included in the study. Throat and nasal specimens were taken two times: on the day of hospital admission (examination I) and on the fourth day after surgery (examination II). Samples were routinely cultured under aerobic conditions. The strains were identified by using API 20E or API 20NE. Susceptibility to selected antimicrobial agents was detected by the disc diffusion method according to NCCLS recommendations.

Results: 24 strains of Gram-negative rods, 17 belonging to Enterobacteriaceae family and 7 -to non-fermenting rods, were found in 28 samples: in 8 patients in examination I (12.7%) and in 16 patients in examination II (25.4%). Double increase of frequency of these bacteria on mucous membranes of nasopharynx in examination II was observed (v 2 test, p = 0.0695). The strains of Enterobacteriaceae were highly susceptible to antimicrobial agents, whereas the most of non-fermenting rods were classified as multidrug resistant organisms. Among the antibiotics used in this study, cefepime and ticarcillin/clavulonic acid were the most active in vitro against the isolated Gram-negative rods. We found that 1 strain produced extended-spectrum b-lactamases and 4 strainsinducible b-lactamases. Conclusion: During hospitalization, in patients with lung cancer undergoing thoracic surgery and preoperative prophylaxis, qualitative and quantitative changes in Gram-negative rods colonizing nasopharynx were observed. In the samples taken on the 4th day after surgery increased prevalence of non-fermenting rods (mostly multidrug resistant) was found. Obtained results confirm the need to conduct analyses of microflora of upper respiratory tract in patients with lung cancer to monitor potential etiologic factors in nosocomial respiratory tract infections.

Outbreaks of Serratia marcescens bacteriuria in a neurosurgical intensive care unit of a tertiary care teaching hospital: a clinical, epidemiologic, and laboratory perspective

Objectives: The aims of this study were (1) to identify the risk factors associated with the development of Serratia marcescens bacteriuria in neurosurgical intensive care units (NSICU) (2) to genotype the pathogens in order to determine a source of infection (3) Environmental surveillance showed the handling of urine jugs to be a point source of contamination. Genotyping and an antibiogram of 14 patients were the same except for two patients.

Conclusions: The patient-related risk factors were identified, and a rapid identification of the organism was made. Altering the method for handling urine jugs provided a focus for the heightened surveillance, infection control measures, and empirical therapy, terminating outbreaks.

Spondylodiscitis after facet joint steroid injection M. Falagas, I. Bliziotis, A. Mavrogenis, D. Antoniou, P. Papagelopoulos (Athens, GR)

Background: Reported infections following facet joint injections are rare and include paraspinal and epidural abscess formation and septic facet joint arthritis. Methods: We describe a case of Pseudomonas aeruginosa spondylodiscitis after facet joint steroid injection.

Results: A 78-year-old male was presented to his local orthopedic surgeon for low back pain. The patient had no neurologic deficits, and with the presumptive diagnosis of degenerative spondylosis, he has been treated with non-steroidal anti-inflammatory agents and physiotherapy for 3 months. Three months later, the patient had worsening of his back pain. Plain radiographs, computed tomography (CT) and magnetic resonance imaging (MRI) of the lumbar spine showed osteoarthritis of the lower lumbar facet joints. Facet joints injection was done using methyl-prednisolone acetate and bupivacaine hydrochloride 0.5%. The patient had temporary relief of his symptoms for 2 days followed by deterioration of his low back pain and acute onset of low-grade fever. Laboratory investigation revealed increased white blood cell count (16.010/ìl), erythrocyte sedimentation rate (83 mm/1st hour), and C-reactive protein (185 mg/l). Spine infection was suspected and ciprofloxacin was administered (500 mg p.o. q12h) for four weeks. However, this treatment led to minimal improvement of the clinical symptoms. At the time of his admission to our institution, his main signs and symptoms included low back pain and tenderness, and increased body temperature. MRI, fine needle aspiration and cultures of the affected area confirmed the diagnosis of Pseudomonas aeruginosa spondylodiscitis at the L2-L3 and L3-L4 levels. The patient was treated with intravenous administration of amikacin (500 mg q12h) and imipenem plus cilastatin (500 + 500 mg q8h), for four weeks, followed by oral administration of ciprofloxacin for 24 weeks. At the latest follow-up, 6 months after the completion of the antimicrobial therapy, MRI of the lumbar spine showed significant narrowing of the L2-L3 and L3-L4 intervertebral spaces with no signs of active infection.

The association of the facet injection with the deterioration of the patient's clinical condition, in addition to the absence of any obvious hematogenous mechanism of infection, suggests an iatrogenic cause of spondylodiscitis. This iatrogenic complication should be included in the risk-benefit analysis of clinicians. Sterile preparation prior to the procedure should be stressed.

Selected virulence factors and resistance to antimicrobials of nosocomial and carrier S. aureus strains

Objectives: To detect the production of selected virulence factors and the resistance to antistaphylococcal therapeutics of S. aureus strains causing sepsis, wound and joint implants infections and to compare them with the carrier strains. Methods: S. aureus strains were isolated from haemocultures (31 strains), surgery wounds (40 strains), prosthetic hip and knee joints infections (42 strains) of patients of Faculty Hospital, Bratislava, and from nasal swabs of carriers (31 strains). Production of hyaluronidase, alpha, beta, and delta-haemolysisns, adherence to glass surface, polysaccharidic extracellular adhesin (PIA) production (on Congo-red plates), and the dynamics of plasmacoagulase production were tested. Resistance to oxacillin, erythromycin, clindamycin, gentamicin, ciprofloxacin, trimethoprim/sulfamethoxazol, quinupristin/ dalfopristin (Q-D), linezolid (LNZ) and vancomycin (VAN) was estimated by disk diffusion test according to NCCLS. Susceptibility to VAN was confirmed by E-test and PBP2a in methicillin-resistant strains (MRSA) was detected by latex agglutination test.

Results: The comparison of investigated groups of S. aureus strains showed, that all, except 2 patient strains, produced alpha-haemolysin -some of them in combination with beta and delta haemolysins. Plasmacoagulase production till the 1st hour of incubation was detected in the majority of patient strains (45 to 70%), while among the carrier strains only in 30%. The most active were strains isolated from joints prostheses infections -JPI (70%). Adherence to glass surface was detected in 93% of all tested strains. The most intensive production of PIA (+++) was detected in 25% strains from haemocultures and 20% from JPI, but only in 12.5 % strains from wounds and 6.5 % of carrier strains. Only 6 strains were found to be MRSA (2 from haemocultures and 4 from wound infections), and all 6 produced only alpha-toxin and PIA. 7 % of strains (from patient samples only) were resistant to gentamicin. All tested strains were susceptible to VAN, LNZ, and Q-D.

Conclusions: Patient S. aureus strains differ from the carrier strains by a more rapid production of plasmacoagulase. Strains from haemocultures and prosthetic joint infections are characteristic by a more intensive production of PIA, which differentiates them from wound and carrier strain groups. MRSA were found only in the group of haemoculture and wound infection strains. All strains were susceptible to VAN, LNZ, and Q-D.

Investigation of a possible nosocomial urinary tract infection outbreak due to ESBLS producing E. coli Material and Patients: The E. coli strains were isolated from equal number of inpatients, two men and two women, mean aged 75 years old who were hospitalized for infections other than UTI (n : 3) and immunosuppression (n : 1). All but one had pyuria (>50 leucocytes p. f), fever (>38°C) and were receiving extended spectrum antibiotics. Indwelling catheters were present in all four patients. Methods: The resistance phenotype was performed by Kirby Bauer and microdilution methods according to NCCLS guidelines. ESBLS production was confirmed by Double Disk Test (DDT) and E-test : cefepime/cefepime-clavulanic (AB BIODISK, Solna, Sweden). Biotyping was based on Wider II (Francisco SORIA Melguizo, SA) automated system results. The ERIC II PCR was used for the molecular typing of the isolates. Results: Although epidemiological data (time and space clustering) as well as phenotypic data (all strains showed the same resistant phenotype and the same biotype) were consistent with possible clonal spread of the same isolate in all patients, molecular typing revealed that only two out of the four E. coli ESBLS producing strains (isolated from patients who shared the same room) were similar and thus could be associated as part of the same outbreak. The remaining two strains had different molecular patterns.

Conclusion: This study supports the clue that classic microbiological studies (colonial morphology, biotype and resistance phenotype) as well as molecular typing methods are both necessary and must be performed to investigate and reveal an outbreak of infection in a hospital setting. Objectives: Measuring the incidence of nosocomial infections and microbial isolates in an Internal Medicine 23 bed-Unit in a 100-bed hospital. Methods: Data were collected according to the NNIS protocol using a dedicated software from Epinfo program.

Results: From October 2002 to December 2003 (for a 14-month term), 1920 patients were enrolled (55% male and 45% female, median age: 76) for a total of 8530 patient-days; 78% of the patients had one or more comorbidities. The infections were 36 in 33 patients (1.71% of the total cases): 20 Urinary tract infections (UTI), 18 of which were catheter-related, 10 pneumonia, 5 blood stream infections (BSI), 1 of which Central Line Catheter-associated, 1 case of Pseudomembranous colitis. The device-utilization ratios were: 1.2% for CVC with an average catheterization of 7 days (103 central line-days); 19.9% for urinary catheter with an average catheterization of 5 days (1702 urinary catheter-days). A Central Line Catheter-associated BSI developed in 8% of patients exposed to CVC with a central lineassociated BSI rate of 9.7 &. An UTI developed in 6.1% of patients exposed to a urinary catheter with an urinary catheterassociated UTI rate of 10.5 &. One patient died because of nosocomial pneumonia (0.9% attributable mortality), 7 patients died having been diagnosed a nosocomial infection. The microbial etiologies of UTIs were: Gram negative in 11 cases, Gram positive in 10 cases, Candida albicans in 3 cases (4 patients showed two contemporary pathogens isolated); BSIs Staphilococci in 3 cases, Gram negative in 1 case, 1 case of Clinical Sepsis. Conclusions: Our study, comparing NNIS data, shows that nosocomial infections may be a problem also in Hospital Units not usually considered at high risk, probably due to advanced age and multiple comorbidities of the patients admitted. Despite a urinary catheter-utilization ratio within the norm, we observed a high incidence of UTI, which however showed an approximate 10% decrease during the period of our research.

Epidemiological aspects of hospital-acquired pneumonia in intensive care units in the far east of Russia Results: The most numerous group consists from 1377 (29.9%) patients with neurological diseases, 40.8% of them were after 50 years and 7.7% (106) were children. The 73.1% of patients among adult ones were males and 68% of patients among children were boys. Children and age patients with chronic infections, severe conditions were the main groups of risk. There were 580 patients (42.1%) including 19 children with lethal outcome. The main reason of lethal outcome in adults were severe combined and skull-brain traumas (75.9%), acute disturbance of brain blood circulation, and infectious disease (5.5%). The severe combined and skull-brain traumas also have taken the leading positions among children. During last 3 years 15 children (79%) have died of this reason, 10.5% of children have died of the infectious disease, and the others have died of the acute disturbance of brain blood circulation and tumors. The 25% of adults and 47.4% of children died of the traumas during the first hours. About 53% of adults and 86.7% of children spent in the ICU more than 5 days. The main causative pathogens of hospital-acquired pneumonias were E. gergovia, S. aureus, P. aeruginosa. The most strains of E. gergovia was resistant to amikacin, ampicillin, amoxicillin, gentmycin, tobramycin, carbenicillin, cefalotin, cefalexin.The most frequent causative pathogen in children was S. aureus which was resistant to penicillin, ceftazidim. But also it was sensitive to cefobite, oxacillin, amoxicillin, gentomycin, lincomycin, chloramphenicol, erythromycin, doxicyclin. P. aeruginosa takes the second place in the ethiological structure of the causative pathogens of hospital-acquired infections which was resistant to cefalexin, cefalotin, carbenicillin, tetracycline, tobramycin, levoflaxacin.

Conclusions: There are some problems in registration of laboratory results of anaerobic infections which are connected with absence of specialists. Also it leads that there is the large group of undiagnosed infections which complicates treatment and increases the frequency of the lethal outcomes.

Unusual bacteraemia-associated pathogens: a 12-year experience These were as following:

• Achromobacter xylosoxidans, following an interventive cardiologic examination. Outcome: death • Pasteurella multocida, in one patient with COPD and in one patient with myelosclerosis. Outcome of both cases: death • Burkholderia pseudomallei, in a patient traveling from S. Asia, on the grounds of pneumonia caused by the same pathogen. Outcome: favorable • Cardiobacterium hominis, in a patient with endocarditis. Outcome: favorable • Yersinia enterocolitica, in a multi-transfused patient. Outcome: favorable • Beta-haemolytic streptococcus group B, in one patient with endocarditis, in a second patient with lung cancer and in a third patient with diabetes mellitus. Outcome: favorable for the first two cases, death for the third • Beta-haemolytic streptococcus group A, in an intravenous drug-abusing patient. The patient left hospital latently. Outcome: unknown • Serpulina pilosicoli, in a critically ill patient with gastroenteritis.

Outcome: death • Cryptococcus neoformans, in a chronic lymphocyte leukaemia patient, and in a second patient with sarcoidosis. Outcome: death for the first, and favorable for the second patient • Radiobacter rhizobium, in a patient with myelosclerosis. Outcome: death • Pseudomonas testosteroni, in one patient with cardiac valve insufficiency and in a second patient with acute abdomen. Outcome: favorable in the first case, death in the second Given the fact that new pathogens emerge as causative factors of bacteraemia, the microbiology laboratory should be alert and able to detect, isolate and identify them. Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 Methods: Forty-two patients with cystic fibrosis, 21 male and 21 female, with an average age of 26.60 years. 78.6% with pancreatic insufficiency. The patients came from the monographic unit of adults with C.F. The sputum samples were cultured following standard microbiological procedures. Colonies quantification in OFPBL agar, saboureaud chloramphenicol agar, blood agar, chocolate agar, chocolate agar with bacitracin, MacConkey agar, mannitol salt agar and Gram stain was performed. Isolates were identified by conventional test and an automated system (Microscan, Dade-Behring) The antimicrobial susceptibility was determined by an automatic system (Micro-scan) and disk diffusion method according to NCCLS recommendations.

Results: The prevalence of infection by Pseudomonas aeruginosa was 47.6%. It was isolated in 20 patients, 10 females and 10 males, with an average age of 23.75 yrs. The number of Pseudomonas aeruginosa isolated in the 20 patients during the study period was 81. 35% of patients were also colonizated by Aspergillus fumigatus, 30% by S. aureus methicillin sensitive, 15% by S. aureus methicillin resistant and 15% by Haemophillus sp. The antimicrobial susceptibility obtained is shown in the table.

Conclusions: A high frequency of Pseudomonas aeruginosa colonization was found in patients with cystic fibrosis. The 100% of Pseudomonas aeruginosa strains were susceptible to colistin in vitro. The highest resistance rate was shown by gentamicin.

Adhesion to catheters and slime production of Staphylococcus haemolyticus isolated from hospitalised patient occurrence of antibiotic susceptibility

Objectives: Last ten years studies showed participation of Coagulase Negative Staphylococci (CNS) in pathogenicity of many diseases. The reason of growing interest about CNS as the ethiological infectious factors can be seen in increasing number of patients from high risk group and use of the invasing diagnostic and clinical methods. The most exposed to the CNS infections except patients with artificial implants. The best characterized CNS pathogenic factors are ability to tissue colonization, adhesion to the synthetic surfaces and evasion.

There is a fundamental stage in the pathogenicity of CNS infections. These properties are caused by the cell wall ingredients (teichoic acid), and also by integral substances associated with bacterial cell such as extracellular polysaccharide (slime).

The study have been done on S. haemolyticus strains isolated from patient hospitalized on Surgical Unit. Aim of the study was to determine pathogenic traits of S. haemolyticus: slime producing, adhesion to biomaterials, antibiotics susceptibility.

Methods: Slime production has been examinated by means of two methods according Christiansen. Adhesion of S. haemolyticus strains was analysed according to Richards method assessing the value of substrate TTC reduced to isolube red formazan. Susceptibility to antibiotics was determined using the discdiffusion method.

Results: Among 44 S. haemolyticus strains, in the test-tube method, there have been 38% labeled as slime producing and 62% as non-producing. In the plate method at 48% slime production was noticed, while 52% strains did not produce slime. 7% of analysed strains were found to have TTC reduction of 3+, 77% of 2+ and 14% of 1+. Among these 2% of the assessed strains did not reduce TTC.

Conclusions: It is quite significant that all strains which have an ability to slime produce, that was proved by means of two methods (test-tube and plate), show a high level of TTC's reduction to formazan. The analysis of resistance to antibiotics in relation to slime production demonstrated more frequent antibiotic resistance of the slime-producing strains. A detailed study into the mechanism and adhesion factors of CNS staphylococci can help prevent infections caused by these microorganism. Results: Ninety-three and 187 isolates were collected from patients in intensive care units (ICUs) and non-ICU inpatient areas, respectively. The remainder were either from outpatients or data were not available. Susceptibility patterns of E. cloacae isolates showed the highest susceptibility to meropenem (99.6%) followed by imipenem (98.7%), cefepime (95.3%), and tobramycin (94.4%). The susceptibility to ciprofloxacin was 89.7%, whereas susceptibilities to ceftazidime and piperacillin-tazobactam were each below 70%. Meropenem (100%), imipenem (98.6%), and cefepim (93.2%) were also the most active compounds against C. freundii. The susceptibilities to ciprofloxacin and tobramycin were 91.8% and 89.0%, respectively, and again susceptibilities to ceftazidime and piperacillin-tazobactam were each below 70%. Rates of resistance (%) were as follows: E. cloacae -ceftazidime 20.9, cefepime 2.6, imipenem 0.4, meropenem 0.4, piperacillintazobactam 12.0, ciprofloxacin 7.7, and tobramycin 3.0; C. freundii -ceftazidime 31.5, cefepime 2.7, imipenem 0, meropenem 0, piperacillin-tazobactam 17.8, ciprofloxacin 5.5, and tobramycin 5.5. For the two species combined, susceptibilities to ceftazidime and piperacillin-tazobactam were significantly lower (p < 0.05) among isolates from ICU patients (59.1% and 55.9%) than among isolates from non-ICU inpatients (71.1% and 70.1%).

Conclusion: Carbapenems and cefepime seem to be the most active antimicrobial agents against isolates of E. cloacae and C. freundii recovered from patients in hospitals located in Germany, Austria, and Switzerland. Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 bacteria. The results of the present study suggest the risk factors for VIM appearance in an Intensive Care Unit. Material and methods: We identified the first 35 patients (20 male, 15 female), who were admitted in a newly established ICU during 3 month period. Tracheobronchial aspirates and urine cultures, were obtained every day during the first three days, and then, every second day after their admission to the ICU. Identification was performed with the PASCO system used according to the manufacturer's instructions. MICs of antipseudomonal drugs, except carbapenems, were determined with the PASCO microdilutionsystem and applying the criteria prescribed by the NCCLS. Susceptibility to imipenem and meropenem was determined by agar disk diffusion in accordance with NCCLS recommendations. A blaIMP, blaVIM specific product was amplified by PCR with crude DNA extracts using published primers and conditions. Qualitative data were expressed as percentages and compared by chi-square test. We considered p < 0.05 to be statistical significant. The p values are based on 2-tailed test results. Statistic analyses were performed using logistic regression (SPSS). Results: We isolated totally 43 multiresistant strains. 33 of those was P. aeruginosa strains,5 was K. pneumoniae and 5 A. Baumannii. All the P. aeruginosa strains were imipenem resistant, and among those only nine (27%), were blaVIM-2 positive. All the strains of K. pneumoniae and A. Baumannii, were blaVIM-2 negative by PCR. Among the total of 9 blaVIM-2 positive P. aeruginosa isolates collected, 22% were from urines and 78% were from bronchial secretions. Discussion: Risk factors for VIM appearance include: (A) Carbapenems administration (n = 0.002), fact that may be due to the augmentation in selections of the above genes under the press of the specific drug. (B) Administration of several b-lactam derivatives (n = 0.005), aminoglycosides (n = 0.039), and combinations between carbapenems-aminoglycosides (n = 0.026).

The statistical significant results of aminoglycoside's administration, may be due, to the augmentation of selection press (the same integron contain the blaVIM gene and three different aminoglycoside resistance determinants, which carried on mobile gene cassettes (aphA15 aacA29A, aacA29). (C) Long length of ICU stay, is a risk factor for blaVIM-2 positive strains, appearance (n = 0.015). Conclusions: Although our sample represents a pilot study measurement, SAA seems to be an interesting inflammatory marker in brucellosis and can be associated with localized infection and hematopoiesis disorders in terms of anemia. Such findings may explain anemia of brucellosis, involving other cytokine production and modulating bone marrow hematopoetic progenitor cells. Further study is required in order to elucidate this enigmatic complication.

Accessing quality in infection control surveillance faecium was isolated from blood in 6 patients (2 pts died), from urines in 16 pts (11 with a urinary catheter, 5 without catheter), from peritoneal fluid (2 pts), from IV catheter (1 pt), from wounds (2 pts), from drainage tubes (3 pts). All GR-E. faecium infections were hospital-acquired. Multivariate analysis, controlling for time at risk and the Charlson Index of Co morbidity, identified prior exposure to IV 3rdG cephalosporin (OR = 10; 95%CI: 1.5-64; p = 0.02), antimicrobial regimen with a betalactamase inhibitor (OR = 5; 95%CI: 0.9-28; p = 0.07) and urinary catheter (OR =20; 95%CI: 1.3-291; p = 0.03) to be independently predictive for GR-E. faecium infection or carriage. Clinical isolates were also resistant to amoxicillin, macrolides and clindamycin, but susceptible to linezolid, vibramycine and intermediate to aminoglycosides, quinupristin-dalfopristin. All PFGE patterns, but one, were clonally related.

Conclusion: Clonal emergence and diffusion of GR-E. faecium is likely to be a result of antibiotic pressure and cross-transmission. As a result, strict recommendations regarding isolation of the patients with multi-resistant bacterial infections and restrictions in the prescription of broad-spectrum antibiotics have been implemented in our hospital.

Genotypic analysis and occurrence of extended spectrum beta-lactamases ( Introduction: Of the various mechanisms of acquired S. aureus resistance to (b-lactams, resistance due to (b-lactamases is the most prevalent. Alterations in the preexisting penicillin-binding proteins (PBPs), acquisition of a novel PBP insensitive to (blactams can also confer resistance to methicillin and (b-lactams. These changes in the outer membrane of proteins include production of supplement PBP (PBP2a or PBP 2Õ) that are encoded by a chromosomal mec gene (mecA, mecB).

Objective: The study of both resistance mechanisms to a-lactams of nosocomial strains of S. aureus. Methods: Over a period of 3 years 340 strains have been tested. All the tests have been performed with the following methods:

(1) oxascreen agar (Bio-Merieux), a Muller-Hinton agar with NaCl 4% and oxacillin 6 mg/ml, for detection of the resistance to oxacillin. (2) MRSA test (Bio-Merieux), an agglutination test for detection of PBP 2Õ. (3) Cefinase discs (Bio-Merieux), for detection of (b-lactamase production. (4) Etest (Solna-Sweden), for detection of penicillin and oxacillin MICs.

Results: Out of 340 strains of S. aureus, 248 (72.09%) were resistant to (b-lactam antibiotics.Out of the latter, 85 strains (34.27%) have shown resistance, which was due to the production of (b-lactamase. Furthermore,134 strains (54.03%) have shown resistance which was due of the alterations in penicillinbinding proteins. Finally, 29 strains (11.69%) have shown resistance which was due to both mechanisms.

Conclusions: (1) High percentage of nosocomial S. aureus strains was resistant to (b-lactams.

(2) In this study, the resistance mechanism of change of the PBPs proteins to PBP2Õ was the most frequent.

Computerised surveillance of nosocomial infections at a university hospital The results were discussed with the departments during surveillance ward rounds. Work time was evaluated using log-tables and an external quality control was performed during a routine phase for 15 weeks. Results: Nearly complete documentation was achieved by the physicians and/or the medical documentation officers. Type and infections rates of VAP, BSIs and SSIs concerning indicator operations will be given and compared to NRZ data. Also data on work time and personnel costs as well as quality assurance will be presented.

Conclusion: Due to the simple and computerized data assessment, personnel time concerning surveillance is very low compared to the non computer assisted surveillance and showed comparable infection rates with national data.

Prevalence of extended-spectrum beta-lactamases in a university hospital (2) and general ward (5) units. Minimum inhibitory concentrations (MICs) were determined in 471 P. aeruginosa clinical isolates to meropenem, cefepime, amikacin, and ciprofloxacin, by Etest methodology according to manufacturer's instructions. Interpretive criteria used were described by NCCLS document M100-S14. Susceptibility patterns of all isolates from ICUs (309), neutropenic patients (37) and general ward units (125) were determined and described. A chi-square test (Altman, 1999) was applied to identify differences in the susceptibility rates among the three unit types studied. p values below 0.05 were considered significant.

Results: Unit differences among the 471 P. aeruginosa clinical isolates are described. Overall resistance rates (%R) were 50.1% to meropenem, 50.7% to amikacin, 56.9% to ciprofloxacin, and 60.7% to cefepime. Higher resistance rates were observed in ICUs for all antimicrobials, except for amikacin. However, differences among resistance rates in specialized units were only significant for meropenem (p = 0.03).

Conclusions: Elevated resistance rates were observed to all antimicrobials, with higher overall susceptibility to meropenem. Meropenem resistance rates were higher among ICU isolates. This could be due to higher antimicrobial use or biased sample, since clonal spread or overvalued resistant clinical isolates could not be ruled out so far.

Predictors of outcome in patients with blood-stream infections Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 Methods: The antimicrobial susceptibilities to nine antimicrobial agents of 61 clinical isolates of Streptococcus pneumoniae recovered from patients with invasive and other clinically significant pneumococcal infections from 2002 to 2004 were studied using E-test and disk diffusion methods following the NCCLS guidelines. Of the 61 isolates, 25% (n = 15) were from blood, 20% (n = 12) from bronchoalveolar-lavage, 16% (n = 10) from tracheal aspirate, 10% (n = 6) from cerebrospinal fluid, 8% (n = 5) from pleural fluid, 7% (n = 4) from endophthalmitis, 5% (n = 3) from peritoneal fluid, 3% (n = 2) from sinus aspirate, 3% (n = 2) from middle ear fluid, 2% (n = 1) from catheter, 2% (n = 1) from fistula specimen. Serogrouping of the pneumococcal isolates was performed using the Quellung technique (Statens Serum institute, Copenhagen, Denmark). Results: Antibiotic susceptibilities and serotypes were shown in Table. Capsular types of 46 pneumococcal isolates belonged to types 23 (24%), 19(15%), 9(15%), 6(13%), 18(11%), 7(11%), 1(7%) and 3(4%).

Conclusions: Antibiotic resistance was common in pneumococcal isolates in our region. Vaccine formulations can prevent the majority of pneumococcal infections. Objectives: Clostridium difficile is a Gram positive anaerobic spore-forming rod. It is present in intestinal flora, in soil and it circulates in hospitals. C. difficile produces different potential virulence factors: a 308 kDa enterotoxin (toxin A) and a 270 kDa cytotoxin (toxin B). Toxin A is the major cause of enteric disease. Colitis can be determined by endogenous or exogenous causes and antibiotic treatment is the most important endogenous cause. The exogenous origin of the infection is referred to orofaecal contamination due to colonised patients. In 1997 an epidemic took place in a surgery division and this fact led to the formulation of an operative protocol for C. difficile associated infection. The operative protocol starts from CDC guidelines and is based on the enteric isolation of C. difficile. The protocol defines the hygienic procedures to be adopted in case of C. difficile associated infection. The target of the protocol is the prompt identification of the symptomatic patient and the subsequent application of procedures to avoid the pathogen's transmission.

Methods: Since May 1997, a surveillance programme is active for C. difficile associated infection and in 2004 the protocol's review was planned. Case definition: C. difficile associated infection is suspected in a patient with diarrhoea. In this case, the patients is investigated for presence of C. difficile and toxin A in stool: if one or both analyses are positive, the division is alerted to implement the protocol's directions and the epidemiological nurse is alerted too. Abstracts g-control chart showed lack of statistical control with up to 100 control values below the centre line through months. G-contol charts from HH and BBH had several out of control periods. Conclusion: A g-control chart was found to be a valuable statistical tools allowing quick identification of an increasing CNS contaminations rate of blood culture. Sufficient skin disinfection think to bee the most important factor for minimizing blood culture contaminants.

Gastric fluid cultures of neonates (2), lymphoma (7), other hematological malignancies (2). The median duration of isepamicin therapy was 10 (4-21) days in the LC arm. Infection was microbiologically and clinically documented in 33 episodes (28%) and in 7 episodes (6%), respectively. The overall success rate without modification was 50% in SC arm, 49% in the LC arm. The mean time to defervescence was 3.8 + 2.5 days in the SC arm and 4.1 + 3.1 days in the LC arm (p > 0.05). Modification of the initial regimen with antivirals and/or antifungals raised the success rate to 60% in the SC arm, 57% in the LC arm. Without a change in antibiotics, the response rates were 5/7 in the SC arm and 5/6 in the LC arm in patients with single-organism gram-negative bacteraemia. Overall sideeffects were observed in 19 patients (29%) in SC arm, 22 (32%) in the LC arm in the intent to treat analysis. The nephrotoxicity were rare and similar in both arms (4 SC, 2 LC, p = 0.31).

Mortality due to infection with or without hemorrhage occurred in a total of 9 patients (4 SC, 5 LC).

Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 Conclusion: Cefepime combined with a short (four days)course of isepamicin seems to be as effective and safe as a longer duration of isepamicin in febrile high-risk neutropenic patients.

Clinical-evolutive aspects of varicella in immunocompromised patients

Immunocompromised patients represent a high risk group for a large number of viral and bacterial infections, requiring a strict clinical and therapeutic surveillance.

Objectives: The study of clinical-evolutive aspects of varicella in adult immunocompromised patients.

Methods: The authors have studied a group of 24 patients from the Clinic of Infectious Diseases in Timisoara suffering from varicella occurring on known chronic affections, evolving on immunocompromised field. Five of the patients were known to have insulin-dependent diabetes mellitus, 4 patients with chronic renal insufficiency (hemodialysis) 3 patients with chronic lymphatic leukaemia, 2 patients with chronic myelogenous leukaemia, 8 patients with tuberculosis, one patient with breast cancer and one patient with Hodkin disease. The diagnosis of varicella was based on the clinical-evolutive symptoms (fever, general itching rash consisting of maculas, papulas and vesicles, affecting both the skin and the mouth, anal-genital, and conjunctiva mucosa occurring in 3-4 waves) and epidemiological elements (contact with patients suffering from varicella or herpes zoster). All patients followed treatment with acyclovir, 2 g/day for 8-10 days, C vitamin, calcium, antihistamines, sedatives, adequate diet and the treatment of the basic disease.

Results: The duration of the skin rash was of 4 weeks in 18 patients and of 5 weeks in 6 patients; the rash was intensively expressed in 4 waves in 20 patients; the following complications were observed: interstitial pneumonias in 15 patients, acute congestive anginas in 5 patients, mouth Candida in 8 patients, cerebellum ataxia in 3 patients, viral meningitis in 2 patients, bacterial skin over-infections in 10 patients, 1 patient with erysipelas, 4 patients with bacterial conjunctivitis; antibiotic treatment with claritromicine in 5 cases, amoxicillin + clavulanic acid in 4 cases and cotrimoxazol in 6 cases was performed. Purpose: The purpose of this study was to determine and associate PCT and CRP levels in serum of septic patients that were hospitalized in the intensive care unit for the last 10 months in Hippocration General Hospital. Methods: During a 10-month interval, 450 serum specimens were examined which corresponded to 92 I.C.U. patients. The PCT value in serum was determined using the immunoluminometric assay method (LIAISON-BRAHMS PCT) taking as normal values 0.10-0.50 ng/ml. The CRP value in serum was determined using the nephelometric method with high sensitivity reagents (Dade-Behring) taking as normal values 0-5 mg/l. Results: From the 450 samples of 92 patients, 143 (32%) had pathological PCT levels that ranged from 1 to 131 ng/ml and 87% of these septic patients had positive blood cultures. In all 450 serum samples (100%) were found high CRP levels that ranged from 3.0 to over 20.0 mg/l 35 (38%) out of 92 patients with sepsis died. Conclusions: Our study shows that: (1) The CRP levels remained high in serum measurements of the patients. (2) The PCT values showed some fluctuation (low and high). (3) The patients with high PCT serum levels also had positive blood cultures. (4) There was no connection between PCT and CRP serum levels. (5) High PCT levels remaining in the serum for a long time consist a bad prognostic factor for disease outcome. Objectives: To determine the usefulness of procalcitonin (PCT) and neopterin measurement as inflammatory markers in patients diagnosed of chronic obstructive pulmonary disease (COPD). To establish if differences are observed in PCT and neopterin levels during a clinically stable period, during an acute exacerbation, and during the process until resolution, as well as in COPD patients with pneumonia. The results will be analysed taking into consideration the severity and etiology of the COPD exacerbation.

Methodology: A total of eighty-nine patients diagnosed of COPD were included in the study. Patients were grouped as follows: Group 1: COPD patients in stable period (4); Group 2: COPD patients undergoing an acute exacerbation, and Group 3: COPD patients with clinical, radiographic and microbiological diagnosis of pneumonia (16). In addition, a control group of 10 healthy individuals was included. Comparison between the different groups of patients was conducted by the Mann-Whitney test in order to evaluate the usefulness of the determination of PCT and neopterin. PCT was measured by immunoluminometric assay (Lumitest PCT, Brahms Diagnostica) and neopterin was measured by enzyme immunoassay (Neopterin ELISA, IBL) in serum samples.

The PCT basal levels in the control group patients were lower than 1 ng/ml (median 0.35, 5-95 percentiles 0.258-2-223).

The higher values were obtained in COPD patients undergoing pneumonia (median 3.11, 5-95 percentiles 0.158-72-44). The neopterin basal levels in the control group patients were lower than 30 ng/ml (median 0.195, percentiles 0.014-5.334). The higher values of neopterin were also obtained in COPD patients affected of pneumonia. In addition, the PCT levels were higher in COPD exacerbated patients than in stable COPD patients. However, significant differences were only observed when comparing the exacerbated severe COPD patients (p = 0.018) to the stable COPD patients. Conclusion: We can conclude that PCT and neopterin show higher levels in COPD patients presenting pneumonia than in those presenting clinical exacerbation without pneumonia and those in a stable period of the disease. Furthermore, a correlation between PCT and the severity of the COPD is observed.

Pneumocystis jiroveci (carinii) pneumonia in the era of PCP prophylaxis Results: Underlaing diseases of these 30 pts. are: NHL -6 (20%), myeloma -6 (20%), Hodgkin's lymphoma -4 (13%), CLL -4 (13%), acute leukaemia -2 (7%), aplastic anemia -1 (3%), CML -1 (3%), HCL -1 (3%), MDS -1 (1%), myelofibrosis -1 (1%), other -3 (10%). PCP occured in 5 pts. (17%) in the time of diagnosis, in 12 (40%) during treatment, in 3 (10%) in remission and in 10 pts. (33%) during progression of underlaing disease. 29 pts. (97%) were without PCP prophylaxis and 21 (70%) had steroid treatment. 27 pts. (90%) have been treated with HD co-trimoxazole (3 pts died before start) with 67% treatment response. 15 pts. (50%) needed mechanical ventilation. Overall mortality of these 30 pts with PCP was 50%. The need for mechanical ventilation were higher in pts. with steroids in anamnesis (52% vs. 44%), with >20 mg of steroids/day (67% vs. 20%), with CD4 count £0.2 · 10 9 /l or £20% (50% vs. 33%) and with underlaing disease in the stage of new diagnosis or during treatment (60%, 58% vs. 33%, 40%).Mortality was higher in pts. with steroids in anamnesis (57% vs. 33%), with >20 mg of steroids/day (53% vs. 40%), with CD4 £0.2 · 10 9 /l or £20% (56% vs. 33%), with neutrophile count £1 · 10 9 /l (60% vs. 45%), pts. on mechanical ventilation (67% vs. 33%) and pts. with underlaing disease in the stage of new diagnosis or progression (80%, 70% vs. 33%, 0%). We did not find higher mortality and higher need for mechanical ventilation in the group of pts. with CD4/ CD8 count £1. (43% vs. 50%, 14% vs. 55%).

Conclusion: Our retrospective analysis demonstrates very low incidence of PCP in the groups of pts with hematological malignancies where PCP prophylaxis is commonly used (e.g. acute leukaemia). By contrast most of pts. with PCP in our institution were pts. with underlaing disease and treatment where PCP prophylaxis isn't routinely used (NHL, Myeloma, Hodgkin's lymphoma, CLL), but steroids were part of treatment protocols (especially >20 mg/day), CD4 count was £0.2 · 10 9 /l or £20% and underlaing disease was in the stage of new diagnosis or progression. These are pts. who need PCP prophylaxis. Background: Imatinib mesylate (STI571 -Glivec) is a selective inhibitor of the Bcr-abl tyrosine kinase, which deregulated expression is involved in the pathogenesis of chronic myeloid leukaemia (CML). We report a case of peritoneal tuberculosis in a patient with global lymphopenia following a four-month treatment with imatinib for a newly diagnosed CML.

Case report: A 37-year-old Swiss male, who was diagnosed with BCR-ABL positive CML in chronic phase, complained after four months of imatinib therapy of abdominal pain, lack of appetite, lost of weight, nausea. Abdominal CT-scan revealed homogenous hepato-splenomegalia, diffuse infiltration of mesenteric fat and ascitis. Ascitis was inflammatory with predominance of lymphocytes and remained sterile on culture. The symptomatology was attributed to an adverse effect of imatinib and the treatment was then interrupted. One month later, he was admitted because of worsening symptoms. An explorative laparoscopy displayed extensive chronic granulomatous peritonitis, and peritoneal tuberculosis was diagnosed by positive Mycobacterium tuberculosis PCR of peritoneal biopsies. Chest X-ray was strictly normal. At admission, he presented global lymphopenia (CD4 155 cell/mm 3 and CD8 39 cell/mm 3 ).

Discussion: This case raises two points: an atypical presentation of tuberculosis in a non-exposed patient and a global lymphopenia in an HIV-negative patient. To the best of our knowledge, this is the first report of an infection suggestive of immune compromise after imatinib treatment. Imatinib is known to induce cytopenia. Moreover, two recent studies have shown that imatinib inhibits the activation and proliferation of T-cells in vitro and delayed-type hypersensitivity in vivo (Cwynarski et al. 2004 , Dietz et al. 2004 ) suggesting that imatinib might affect immunity and could lead to a higher than expected incidence of opportunistic infections. No epidemiological data are yet available to support this hypothesis. Of note, tuberculosis itself induces transient lymphopenia in HIV-seronegative patients and patients with CML are at risk for infection due to a possible dysfunction of the immune response.

Conclusion: Further studies of imatinib effect on immunity and on the incidence of opportunistic infections should be performed. Checking CD4 and CD8 T cell counts in patients during imatinib treatment could be considered.

Identification of lactic acid bacteria isolated from blood samples and their susceptibility to antibiotics (3 strains), Lactobacillus rhamnosus (7), Lactobacillus paracasei ssp. paracasei (2), Lactobacillus salivarius ssp. salivarius (1), Lactobacillus fermentum (1), Leuconostoc mesenteroides (9), Leuconostoc lactis (5) and Pediococcus pentosaceus (1). The (GTG)5-PCR fingerprinting and whole cell protein profile analysis confirmed above identifications and furthermore identified four strains which were biochemically classified at the genus level as Lactobacillus sakei ssp. Carnosus, Lactobacillus curvatus, Leuconostoc pseudomesenteroides and Weissella confusa. All investigated strains (19) were resistant to vancomycin, teicoplanin and metronidazol and most of them to cotrimoxazol. Susceptibility to cefotaxim, imipenem, gentamicin and ciprofloxacin was variable. Conclusion: Lactobacillus rhamnosus and Leuconostoc mesenteroides were the most common LAB species isolated from blood samples. Although isolation of LAB from blood samples is rare, it is of clinical significance and it should be taken into account during treatment decision -especially when they are isolated in pure culture from sterile sites.

Risk factors for bacterial infections in multiple myeloma treated with vincristine-adriamycindexamethasone schedule A. Zaharof, R. Koutri, G. Svoukas, K. Kanakis, N. Kostakos, C. Flevaris, C. Petrogiannopoulos (Halandri, Athens, GR)

Objective: We evaluated bacterial infections (BIs) in patients with multiple myeloma (MM) treated with vincristine-adriamycin-dexamethasone (VAD) schedule during the last 4 years in our Department. Design and methods: Sixty-five patients were studied during 290 VAD cycles. VAD was given by continuous intravenous infusion (CII) to hospitalized patients. The characteristics of patients and VAD schedules were retrospectively analysed to detect correlations with the incidence of BI. Results: By analyzing each VAD cycle, we found that profound hypogammaglobulinemia (p = 0.05) and post-treatment neutropenia (p = 0.09) were associated with a higher risk of infection, while renal function impairment was significantly correlated with BI risk at multivariate (p < 0.001) analysis. We evaluated only the first 3 months of therapy, characterized by a significantly higher incidence of BI than the later period (p < 0.0001).Continuous schedule (p = 0.07), and profound hypogammaglobulinemia (p = 0.01) were associated with a tendency to a higher risk of infection. The high probability of CII-related infection was demonstrated to depend on the frequent development of nosocomial infections. Conclusion: Patients with profound hypogammaglobulinemia who receive VAD as first line treatment are at a major risk of BI up to the completion of the third month of therapy. These patients require antibacterial prophylaxis with intravenous immunoglobulins which could be appropriate and effective. Objectives: M. Fuerth was described in 2002 as a member of the rapid growing Mycobacterium chelonae complex. Its clinical significance and optimal therapeutical regimens have still remained unclear.

Methods: Here we report on a case of a 39 years old woman with catheter related disseminated infection due to M. Fuerth following bone marrow transplantation due to aplastic anemia. Results: M. Fuerth could be cultivated in April 2004 from two blood cultures and in high numbers from a central venous catheter. Therapy was started using imipenem and clarithromycin. During the following 4 months, no signs of infection could be observed, but the patient developed disseminated purple coloured infiltrations of her the skin, which were initially regarded as cutaneous GvHD. In September 2004, a skin biopsy revealed large amounts of mycobacteria, which could also be subsequently identified as M. Fuerth. According to susceptibility testing results, a therapy with linezolid (Zyvoxidä) was started.

During the next three weeks, the lesions improved and were sterile upon follow-up biopsy and culture. Ultimately, the patient succumbed due to chronic transplant-associated complications.

Conclusion: As other members of the rapid growing mycobacteria, M. Fuerth is able to cause catheter-related and disseminated infections in severely immunocompromised patients. Linezolid, which has in vitro activity against many mycobacteria was effective led to resulting in rapid improvement of the mycobacterial skin infiltrations and sterilization of blood cultures and skin biopsies.

Massive pericardial effusion due to Salmonella enteritidis in a patient with rheumatoid arthritis A. Cefle, S. Gundes, B. Erkol, N. Dagdelen (Kocaeli, TR)

Objectives: Although pericardial involvement is frequent in patients with rheumatoid arthritis (RA), it is rarely symptomatic and the course is mostly benign and self-limiting; also, septic pericarditis is extremely uncommon. In this case report, we describe a patient with RA who developed massive pericardial effusion due to Salmonella. Methods: The patient, a 48-year old man who suffered RA for two years, was on low-dose corticosteroid and methotrexate treatment for the last four months. He presented with palpitations and retrosternal pain. On physical examination he was not in cardiopulmonary distress. Erythrocyte sedimentation rate was 80 mm/h and the leukocyte count was 11300/mm 3 . An echocardigraphic examination revealed minimal pericardial fluid collection which was considered to be due to RA and the dose of corticosteroid was increased. However, the amount of fluid increased and periods of fever (38°C) were observed. A pericardiocentesis was performed and 600 mls of purulent fluid was drained. The pericardial fluid cultivated to BACTEC system were passaged to Endo and Blood agar plates. Conclusions: Our study suggests that high percentage (30%) of vaccinated children are non-immune after 5 years and need the revaccination.

Serotypes of Salmonella isolated strains in northern Greece and antimicrobial resistance rates A. Poulou, F. Markou, P. Rozi (Serres, GR)

Objectives: Salmonella enterica is one of the main causes of diarrhoea world wide. In this study, we present the serotypes of Salmonella isolates from diarrheal patients over 2 years in our hospital in Northern Greece and determine the antimicrobial resistance rates.

Methods: Over a 2-year period a total of 648 stool specimens from diarrheal patients were cultured. Identification of the isolated strains was made by conventional microbiological methods while determination of their antibiotic susceptibility against ampicillin (AMP), ampicillin/sulbactam (SAM), tetracycline (Te), chloramphenicol (Cl), ciprofloxacin (Cip), ceftriaxone (CRO) and trim-ethoprim-sulfamethoxazole (SXT) was performed using Auto Scan 4 of Dade Boehring. The disc diffusion method of Kirby Bauer was used when necessary according to the NCCLS recommendations. Serotyping was performed with monospecific antisera by the slide agglutination method.

Results: A total of 53 Salmonella strains were isolated, 11 of them (20.8%) derived from adults and the majority 42(79.2%) from children. There is also a seasonable distribution of Salmonella cases with a peak in the summer months. The most prevalent serotype was S. enteritidis 9:g,m (75.5%), followed by S. typhimurium 4,5:i (18.8%), S. goldcoast 6.8:r:1,w(1.9%), S. blockley 6.8:k:1,s (1.9%) and S. thompson 6,7,14:k:1,s(1.9%). 49 strains (92.4%) were susceptible to all antibiotics tested while three strains (5.7%) 2 S. typhimurium and 1 S. goldcoast were resistant to AMP, SAM, CL, Te and one strain of S. enteritidis (1.9%) was resistant to Te. Medical case histories revealed two interesting incidences of family infection. In one, S. enteritidis was isolated from five members of the same family who had eaten infected meat. In the second case, S. typhimurium was isolated from two infants after close contact with their grandfather, who was found to be a chtonic carrier of the same strain.

Conclusions: Salmonella enteritidis predominates over the other serotypes. Children are more susceptible than adults to salmonellosis. Although in our region the resistance rates are low in comparison with other countries the rational use of antibiotics and continuous surveillance are essential in order to prevent the dissemination of resistant Salmonella strains. Objective: The opioids including morphine are believed to be responsible for many systemic effects in drug abusers. Involving immune system the opioids increased susceptibility to infections. The effect of acute administration of morphine on Systemic HSV-1 infection was evaluated in this study. Method: HSV-1 was propagated and titered on BK cell line. Three groups of BALB/c mice were received 50 mg/kg, 75 mg/kg or PBS (positive control) subcutaneously, 24 h after ip injection of 2 · 10 7 TCID50 HSV-1. Under the same conditions a group of mice just received 75 mg/kg morphine as negative control. The survival rate was followed for 14 days. The cytotoxic activity of NK cells using MTT assay.

Results: Survival analysis demonstrated that comparing to control group administration of 75 mg morphine significantly reduces innate immunity to hsv-1 infection. The NK cell activity has been reduced in morphine-treated group in comparison with saline-treated one.

The results showed that adverse effects of Morphine on innate immunity can be taken as a result in enhanced rate of mortality in HSV-1 infected mice. Objectives: Disorders of polymorphonuclear leukocytes (PMNL) phagocytoses and intracellular killing activities play vital role in pathogenesis of diabetic foot infections. High blood levels of interleukin-1 beta (IL-1 beta), which is a proinflammatary cytokine, are detected at the initial phase of inflammations. In this study, we aimed to investigate the changes in the phagocytic and intracellular killing activities of PMNL and blood levels of IL-1 beta during the treatment of diabetic foot infections (DFI ) were obtained in the pretreatment period and at 2nd and 4th weeks of the treatment period. Polymorphonuclear leukocytes (1 · 107 cells/ml) were separated from venous blood with EDTA by Ficoll-Hypaque gradient centrifugation. These PMNL were incubated with Candida albicans blastospores and number of ingested organisms per 100 PMNL were calculated. Wound care, rational antibacterial therapy and hiperbaric oxygen therapy constituted the treatment protocol. The IL-1 beta levels were detected in the three period by ELISA (Quantikine HS, R&D Systems, Inc., US). Statistical analysis was performed by Mann-Whitney U-test and p < 0.05 was admitted to be meaningful.

Results: Twenty-three cases responded to the therapy completely. Amputation was performed to eight of 15 cases who did not respond to the therapy. A statistically significant increase in phagocytic activities of PMNL were detected only in responder group, whereas no significant change in intracellular killing activities of PMNL due to treatment were detected in none of the groups. A statistically significant decrease in the blood levels of IL-1 beta was detected in responder group at the 2nd week. In non-responder group, IL-1 beta levels increased slightly but it was not statistically significant. At the 4th weeks of the treatment, the IL-1 beta levels decreased slightly in both groups but it was not statistically significant. On the other hand, there was significantly change in IL-1 beta level between responder and non-responder groups in 2nd weeks of the treatment (1.97 ± 3.96 vs 4.91 ± 6.88, p = 0.025).

The present study confirmed that increased phagocytic activity of PMNL and decreased blood levels of IL-1 beta correlate well with good clinical prognosis in the treatment of DFI.

Chlamydia pneumoniae and risk factors of acute myocardial infarction Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 Conclusions: We believe that C. pneumoniae's property of developing chronic infection triggers processing of other AMI risk factors although it is not a direct risk factor for AMI.

Pre-and post-operative changes in complement C3, complement C4 levels in acute appendicitis Objectives: Cytokines and nitric oxide play an important role in the human immunological response. The aim of study to determine their role in immune response in patients infected with Echinococcus granulosus. Methods: Thirty-two patients with cystic echinocosis (CE) were studied and all underwent surgery. Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin IL-1beta, receptor of soluble IL-2 (sIL-2R), IL-6, IL-8, nitrate/nitrate, and C-reactive protein (CRP) were evaluated before surgery, on first day, and three months post operation. Data were compared with those obtained from 30 healthy volunteers. Results: IL-6 was elevated in vast majority of CE patients (96.9%). IL-8 was increased in 12/32 (37.5%). Slight increases of sIL-2R, and TNF-alpha levels were found in limited number of them particularly those showing cysts in the central area of the liver (5/32, 6/32). IL-1 beta level was not elevated in any patient. CRP and nitrate /nitrate levels were also increased. A positive correlation between CRP and IL-6 (r = 0.78, p < 0.001) was found confirming the link between inflammation due to CE and activation of monocytes. All patients were completely recovered and studied parameters levels were declined to normal levels except one patient in whom recurrent disease occurred at interval of 2 years from the first operation. Conclusion: These results suggest that there are different immunoregulatory events and cytokines response during CE and may be in part related to slight monocytosis in CE patients. IL-6, NO and CRP were involved in the host immune response against E. granulosus and may be useful markers in diagnosis, monitoring CE management and evaluation surgical stress.

Detection of Fc receptors released by streptococcal isolates of anginosus group G. Bancescu, R. Matre, N. Skaug, A. Bancescu (Bucharest, RO; Bergen, N)

Objectives: Oral streptococci, especially those belonging to the anginosus group, are the main facultatively anaerobic microorganisms involved in oral infections. The aim of the study was to detect soluble Fc receptors (FcR) secreted by some streptococcal strains of anginosus group isolated (either as single bacteria or in association with other microorganisms) in pus samples collected from patients with different pyogenic oral and maxillofacial infections.

Methods: The strains were identified at species level using the Rapid ID 32 STREP system (Bio-Merieux, France). Cell-free supernatant samples obtained from the culture of each isolate were blotted onto nitrocellulose membrane (Bio-Rad Laboratories) using a multifiltration apparatus. Horseradish peroxidaserabbit anti-peroxidase complexes (PAP) (DAKO-Immunoglobulins a/s, Dakopatts, Denmark) were used to react with the bound FcR and afterwards, with the second antibody (goat antirabbit antibodies conjugated with horseradish peroxidase). In addition, 4-chloro-1-naphthol (Sigma Immuno Chemicals) was used to detect horseradish peroxidase in order to visualize the immune complexes.

Results: Forty isolates were identified as S. anginosus, while only one and three isolates were found to belong to S. intermedius and S. constellatus species, respectively. Positive FcR reactions (grey-blue dots) were obtained in all test samples. Conclusion: Streptococci of anginosus group involved in pyogenic infections secrete small amounts of FcR which can be detected by this sensitive dot-immunobinding assay.

Pathogenesis, animal models including experimental treatment Objective: Microglial cells behave as cells of innate immunity in the brain. In order to study the pathogenesis of meningitis blood monocytes were triggered by cerebrospinal fluid (CSF) and by sera from patients with bacterial and abacterial meningitis Methods: Cerebrospinal fluid and blood serum were collected upon admission from nine patients with bacterial meningitis and twenty-four patients with abacterial meningitis. Mononuclear cells were isolated from healthy individuals after density gradient centrifugation of heparinized whole blood over Ficoll-Hypaque. They were then washed three times with Phospate-Buffered Saline (pH: 7.2) and suspended in flasks. After incubation for one hour at 37°C under 5% CO 2 , nonadherent cells were removed and monocytes were re-suspended in 12-well plates in RPMI with 10% FBS and 2 mM glutamine at a density of 1 · 10 5 cells/well. After 30 minutes of incubation at 37°C in 5% CO 2 100 ll of sampled CSF and 100 ll of sampled serum of each patient were added into different wells of each of the volunteers. Cells were subsequently incubated for 18 hours at 37°C in 5% CO 2 . TNF-a, interleukin-1b (IL-1b), IL-10, IL-12, IL-6 and interferon-c (IFN-c) were measured in culture supernatants with an enzyme immunoassay; procalcitonin (PCT) by an assay based on immunochemoluminescence. Objectives: The aim of the study was: 1/to evaluate the influence of isolated from patients with gastric ulcers/GU/and chronic gastropathy/CG/Candida strains, on gastric secretion and gastric ulcer healing in rats in conditions of Aspirin/ASA/ oral administration. 2/to evaluate the influence of probiotics on GU healing. Methods: We investigated 60 Wistar rats equipped with gastric fistula, with GU induced by acetic acid/ulcer area 28 mm 2 /. Animals were divided into 3 groups. I: rats inoculation for 25 days with C. albicans: 105 CFU/ml/i.g. was preceded by 5 days supply of proteinase 0.2 U/kg i.g., promoting fungal colonization and aspirin/ASA/: 40 mg/kg/i.g. II: Rats received Candida ASA and proteinase as group I, besides Lactobacillus acidophilus 106 CFU/ml i.g. III: rats with GU-induced, given vehicle/saline/and proteinase. Animals were sacrificed after 4, 15, 25 days upon induction. The ulcer area was measured with planimetry, the gastric blood flow/GBF/was determined H 2 gas clearance. Gastric biopsy was taken for quantitative and qualitative mycological investigation and for the histopathology. Plasma levels of IL-1b, TNF-a, gastrin were measured. RT-PCR expression of IL-b, TNF-a was evaluated in gastric tissue. Results: In rats inoculated with fungi, given ASA, gastric output was reduced in over 40% and GBF was decreased ,in comparison with rats given vehicle/group III/. The rats from group III showed progressive decrease in the area of GU by 13%, 45% and 95% at 4, 15 and 25 day, respectively. In contrast , the ulcers were present till 25 day in all rats inoculated with Candida . In rats given probiotic, decrease of GU area was observed more rapidly, in comparison with group I. In Candida colonized rats, upregulation of TNF-a, IL-b mRNA and the rise of plasmagastrin, TNF-a, IL-b, IF-c levels was recorded. Conclusions: 1. Fungal colonization could be achieved in rats by antisecretory or ASA treatment. 2. Fungal infection markedly reduces gastric secretion, delays gastric ulcer healing, probably due to fail of GBF at ulcer area and maintaining the mucosal inflammation, involving expression and release of IL-b, TNF-a. 3. Administration of probiotic reduces the delay of GU healing caused by Candida inoculation. Introduction: Helicobacter pylori are commonly associated with gastritis but only sometimes it causes clinically significant infectious diseases such as gastric and peptic ulcer diseases. The development of disease depends on the virulence of the infecting H. pylori strain, the susceptibility of the host, and environmental co-factor. The cytotoxin associated protein encoded by cagA gene is an important virulence factor that is produced by some H. pylori strains, and has been used as virulence marker in some populations. The aim of study was to determine the prevalence of cagA gene as a virulence factor on H. pylori strain types in Iranian GUD and PUD patients. Shigella sonnei is a major cause for diarrheal disease in developed as well as in developing countries. Epidemiologic studies of this organism have been limited by the lack of a simple and effective method for comparing strains. The study of epidemiologic markers is important in an attempt to trace the sources of infection. We used PFGE, ERIC-PCR to study the genetic relatedness of 78 isolates of S. sonnei collected in Seoul. For quantitative illustration of the relationships within a large body of isolates, computer generated dendrogram were used to determine the number of isolates in pulsotypes at Dice coefficients of similarity of 80%. From 2000 to 2003, 78 isolates of Shigella sonnei were isolated from patients in Seoul. By the PFGE experiment, Over 80% band similarity has divided 6 Group(A~F) band patterns. XbaI digestion produced about 40 fragments and their sizes ranged from 32.4 to 582 kb. Isolated from the same outbreak showed identical PFGE patterns, Most of the different outbreak strains and sporadic strains showed identical or different PFGE patterns. The ERIC-PCR technique successfully typed all isolates examined and produced bands in the 300 to 2,000 bp size range. The susceptible strains to Chloramphenicol, Amikacin and Cefoxitin was the most prevalent 100%, respectively. In case of the multiple antibiotic resistant pattern, the most prevalent pattern was NA-SM-SXT-TE, followed by AM-NA-SM-SXT-TIC-TE. During an outbreak of infection, the aim of bacterial typing is to provide laboratory evidence that epidemiologically related isolates are also genetically related. Isolated from the same outbreak showed identical PFGE patterns, Most of the different outbreak strains and sporadic strains showed identical or different PFGE patterns. We found that PFGE and ERIC-PCR have the highest discriminatory power for differentiation of strains of S. sonnei. ERIC-PCR fingerprinting is particularly useful because of its simplicity and represents a less time consuming procedure. We conclude that it is possible for a typical clinical laboratory to analyze a large amount of PFGE information on Shigella isolates obtained under controlled conditions. Such data analysis should enhance surveillance capabilities and give indications of further work to various aspects of bacterial pathogenicity of the species.

Inactivation of mraZ and mraW genes of dcw cluster in Salmonella enterica serovar Typhimurium SL1344

The versatility and adaptability of bacterial pathogens as well as complexity and variety of antimicrobial resistance mechanisms even against last generation antibiotics actually in use, are causing a serious health problem in developing countries with re-emerging of infectious diseases considered totally eradicated. Our working group is trying to discover a new antibacterial target highly specificic, selective and essential for survival of our pathogenic bacterial study model: Salmonella enterica serovar Typhimuruium SL1344 (STM). It presents as an important virulence factor its capacity to replicate intracellularly in epithelial cells, been necessary to develop a very complex molecular mechanism to improve penetration, intracellular survival and replication in the host cell. Focusing bacterial division process and specifically the mraZ and mraW genes, located at the beginning of the division cell wall cluster, we are trying to determine the potential character of this genes like a useful target in design of therapeutic strategies that will allow to limit infection and avoid the acquisition of antimicrobial resistance mechanisms. Since the pathogenicity of STM is given by its capacity to penetrate to non-phagocitic host cells, we have designed a deletion mutant of STM for the mraZ and mraW genes implied in the process of cellular division. This mraZWmutant was obtained by using a very simple PCR and homologous recombination technique. We first used a set of 60 nt-primers to amplify the chloramphenicol resistant gene flanked by FRT regions (FLP Recognition Target) followed by homologous regions of genes adjacent to mraZ and mraW genes. We then used the PCR product to transform competent STM cells containing the low number copies and temperature sensitive pKD46 vector, which express the red recombinase of phage lambda under the control of arabinose inducible promoter. Afterwards, we selected the chloramphenicol resistant mutant. Finally, this antibiotic resistance gene was eliminated using the pCP20 plasmid that shows ampicillin and chloramphenicol resistance, temperature-sensitive replication and thermal induction of a recombinase FLP synthesis. We have subjected this mutant to several tests like the in vitro intracellular infection test using non-phagocitics cell-lines: HeLa, NIH/ 3T3 and NRK. In parallel, an Ôin vivoÕ test using a mouse model in order to determine the kinetic value of intracellular proliferation has been carried out.

Identification of the pathogens caused the outbreak of Streptococcus suis toxic shock syndrome and meningocephalitis of swine and human J.Q. Tang, C.J. Wang, H.B. Guo, X.Z. Pan (Nanjing, RC)

Objectives: To reveal the relationship between the pathogens of toxic shock syndrome and meningocephalitis outbreak of pigs and human in 1998 in East China.

Methods: Epidemiological survey, isolation and identification of pathogenic bacteria were performed. Results: Characters of the disease were quick onset, serious symptoms, short course and high mortality. The main clinical manifestations of the disease were high fever, sometimes with nausea, vomiting and diarrhoea, then might developed to myositis, fascitis, DIC, multiple organ failure, shock, and usually died in 2-3 days. Among 25 patients, 16 manifested clinically as streptococcal toxic shock syndromes (STSS) and 9 streptococcal meningocephalitis syndrome, with mortality 81.25% and 11.11%, respectively. 16 bacterium strains isolated from blood and cerebrospinal fluid specimens from the diseased pigs and patients were identified as Streptococcus suis based on biochemical reactions and 16S rRNA gene sequence analysis. The isolates from both pig and human also showed indistinguishable antibiotic and pulse-field gel electrophoresis patterns. Conclusion: The pathogen isolated from the blood of patients and pigs were identified as the swine streptococci. It suggests that the outbreak of Streptococcus suis sepsis spreads from pigs to humans.

Real-time PCR screening of women with termruptured membranes for carriage of group B streptococcus Objectives: To evaluate a Light Cycler PCR assay for the detection of group B streptococcus (GBS) carriage in women with term-ruptured membranes in comparison with conventional culture.

Methods: Low vaginal swabs were obtained from 143 pregnant women who presented with term rupture of membranes. After incubation for 1 h in brain-heart infusion broth, DNA was extracted with the InstaGene matrix (BioRad), and real-time PCR for amplification and detection of the cfb target was performed with the LightCycler (Roche). Samples were processed in parallel by conventional culture on HSA and ISLAM plates, with confirmation of GBS isolates by latex agglutination. Results: Twenty (14.0%) samples were positive for GBS by conventional culture, whereas the PCR assay detected GBS carriage in only 10(7.0%) cases, although two PCR-negative samples were positive on repeat testing. The PCR false-negative results did not always correlate with low numbers of target cells, thereby indicating a problem with extraction and/or inhibition rather than sensitivity per se. The negative and positive predictive values of the PCR assay were 92.5% and 100%, respectively, but the overall sensitivity of the assay was only 50%.

Conclusions: Although the real-time PCR assay generated a result within 3 h of receipt of a sample, the current method highlighted the need to further optimise DNA extraction and purification protocols for use in the clinical setting. Further studies will continue to address the optimisation of the sample processing and DNA extraction steps.

Today's medicine takes advantage of the latest achievements in natural sciences and successfully employs new techniques to the diagnostics and treatment of diseases. Recently moleculargenetic science has been applied in the field of diagnostics of sexually transmitted diseases (STD) in addition to the conventional culture-based and serological methods: the technology of in vitro specific DNA-amplification or Polymerase Chain Reaction (PCR). The molecular methods have many advantages: high sensitivity, high specificity, option for standardization and Human immunodeficiency virus (HIV) is a member of the Retroviridae family. The HIV genome resembles that of other retroviruses with the typical gag, pol, and env gene organization. HIV establishes a permanent infection once the proviral DNA integrates into the host cell genome. HIV has been shown to infect a variety of cells, including peripheral lymphocytes, macrophages and other CD4+ cells. HIV is associated with immune system dysfunctions and, is responsible for opportunistic infections involved in AIDS. Transmission of HIV through blood transfusion and diagnosis of infection in hospitals and public health settings continues to be a worldwide concern. Polyclonal, mono-specific antibody against HIV recombinant p24 antigen, the capsid protein region, has been developed in rabbit and purified with gel filteration and affinity chromatography techniques. The purified antibody has been conjugated with the horseradish peroxidase enzyme and a capture enzyme linked immunosorbent assay (ELISA) has been developed for detection of p24 Antigenemia. HIV-negative human sera failed to react with the developed assay, thereby showing the specificity of the HIV antigenaemia assay. Seventy-five human positive serum samples as well as human negative serum samples were tested for the presence of p24 antigen by the developed and a reference assay (Abbot Lab., USA). There was a 100% concordance when the samples tested with both assays. The results of this study showed that the developed p24 antigenaemia assay could be used for detection of HIV infection in human early before appearance of any detectable antibodies against the virus. This assay appears to be highly sensitive and specific to HIV. The test also has several advantages, including a relatively short test time and, most importantly, an adequate and reproducible supply of antibody to be used. Further research is needed to better evaluate the developed assay reactivity in a larger number of HIV positive sera.

The Objectives: The aim of this study was to determine procalcitonin (PCT) and C-reactive protein (CRP) levels in early syphilis (ES) cases and evaluate these indicators in diagnosis of syphilis. Methods: Twenty-nine patients with ES, 30 patients with bacteraemia and 30 healthy blood donors (control group) were included in this study. PCT levels in blood samples were measured by monoclonal immunoluminometric method. Results: The mean PCT levels (minimum-maximum) were found 0.80 (0.55-1.680) ng/ml in ES group, 7.5 (0.68-35.4) ng/ml in bacteraemia group, and 39 (0.15-1.25) ng/ml in control group, The PCT levels in the ES cases were statistically higher than those observed in healthy blood donors (p < 0.01).

The area under the ROC (receiver operating characteristic) curve using PCT to predict early syphilis was 0.920 (95% CI: 0.831-1.008). The sensitivity, specificity, positive predictive value and negative predictive value for PCT were 100%, 90%, 90%, and 100%, respectively at 0.5 ng/ml, which was an optimal concentration. Consequently, PCT levels were found slightly higher than the cut-off value (mean: 0.80 ng/ml) in ES.

In conclusion, this research is the first preliminary study for determination of PCT levels in ES cases in spite of few number of case-control cases. Our results suggested that PCT is a useful and additional marker at 0.5 ng/ml optimal serum concentration according to the ROC curve when conventional laboratory test results were false positive and false negative, Nevertheless, we believe that new, extended serial case-control studies are needed to further support our results.

The microagglutination test as a complement to ELISA examination in serological diagnosis of Legionella infections K.W. Pancer, W. Gut, H. Stypulkowska-Misiurewicz (Warsaw, PL)

In Poland, laboratory diagnosis of Legionella infections generally is based on serological investigation. Bacteria L. pneumophila sg 1 are more frequent cause of severe pneumonia, less frequent are L. pneumophila other serogroups or other Legionella species. Examination of serum sample using ELISA IgA, IgM and IgG assays is the method of choice in our laboratory for detection L. pneumophila sg 1. For L. pneumophila non-1 or other Legionella species infection the microagglutination test has been used.In our study we determined the significant titre of serum antibody to some L. pneumophila non-1 serogroups and other Legionella species on the base of results of examinations of serum samples collected from blood donors. The titre of antibody was determined using 14 antigens prepared in-house from the reference strains L. pneumophila and others Legionella. Statistical analysis of obtained results allowed to determine cut-off titre for all used antigens. The value of cut-off was 64 for L. pneumophila sg 1, 2, 4, 5, L. longbachae, cut-off = 128 for L. pneumophila sg 3, 7, 8, L. micdadei, L. jordanis, and cut-off = 256 for L. pneumophila sg 6, 12, L. bozemanii, L. anisa. Using those cut-off points we are able to eliminate not significant titre due to some cross-reactivity of L. pneumophila serogroups.Determination of specific serum IgA, IgM and IgG antibody levels to L. pneumophila sg 1 by commercial ELISA is more sensitive, standarised, informative and faster than microagglutination test. However, from our point of view the microagglutination test should be used as complement test to ELISA, especially for diagnosis of infection due to L. pneumophila non-1 serogroups or other Legionella species. In 16% of all serum samples tested this year the significant titre of antibody specific to one of serogroup of L. pneumophila non-1 was found using MAT. In those sera increased but not significant level of IgM antibodies to L. pneumophila sg 1 (in range 0.32-0.75) or IgA antibodies (0.38-0.53) was determined by ELISA. It is probably connected with crossreactivity of some antigenic structures of L. pneumophila strains. Moreover, two cases of legionellosis due to L. micdadei were diagnosed. In those cases the antibiotic had been changed and the patients recovered. Because of unspecific symptoms and rather high mortality rate in legionnairesÕ disease the microbiological diagnosis of Legionella infection should be done using as many supplementary techniques as possible. Objective: The aim of this study was to evaluate external proficiency testing in microbiology laboratories located in Tehran and suburbs for identification of three unknown bacteria.

Methods: The 14th survey of external programme for 2004 distributed to 532 laboratories in Tehran. The selected microorganisms were: Shigella flexneri, Sreptococcus agalactiae and Stenotrophomonas maltophilia. S. flexneri were sent to all 532, S. agalactia to 395, and S. maltophilia to 137 microbiology laboratories. We asked all laboratories for identification each unknown organism, performance of susceptibility testing for S. flexneri and return their answer to reference laboratory within 2 weeks receipt of samples. Scoring of results determined according to WHO external surveys criteria.

Results: Of 532 laboratories we received answer from 428 (80.5%) laboratories. Of 428 laboratories 205 (38.5%) produced correct answer for identification of S. flexneri and received 3 score of points. and 31(5.8%) laboratories produced incorrect answer for identification of this organism. The remaining 192 (36.1%) laboratories received 1-2 score of points (mean score 2.2, SD ± 0.91). Of 137 labs only 11(8%) labs produced correct answer for identification of S. maltophilia and 66 (48.2%) labs received zero score of points. The remaining labs 23 (16.8%) received 1-2.5 score of points (mean score 81, SD ± 1.13). Of 395 labs 146 (37%) identified S. agalactiae correctly and obtained 3 score of points and 32 (8.15) were not able to identify this organism and therefore received 0 score of points The reaming laboratories 137( 36%) obtained 1-2.5 score of points (mean sore 2, SD ± 1.1). The results of susceptibility testing were satisfied and the majority of laboratories produced correct answer for susceptibility testing of S. flexneri (mean score 4.52). It is assumed that the result of external quality control in Tehran microbiology laboratories was not acceptable. Conclusion: Our study reveals that unfortunately proficiency testing in microbiology laboratories of Tehran is poor .The poor results were due to: defective reagents, l culture media inappropriate settlement of internal quality control programme and of unqualified technologists. Methods: In an analytic study 232 patients with gastrointestinal signs were studied. Endocopy was done and 2-3 biopsies were taken from antrum. Specimens were processed by shandon tissue processor (citadel) and prepared sections were stained by Hematoxylin-Eosin and Giemsa. Slides were studied by two pathologist and one microbiologist. Also 5 cc blood was taken to determine anti HP (IgG) by ELISA method. Objective: Campylobacter spp. are recognized as a major cause of acute bacterial enteritis in the developed countries. Several potential virulence factors have been proposed as important factors in the pathogenesis of Campylobacter jejuni e.g. motility, adherence, invasion and toxin production. The only toxin of C. jejuni characterized at the molecular level is the cytolethal distending toxin (CDT); cdt genes have also been found in various other Gram negative bacteria. The aim of this study was to examine whether the level of CDT expressed had any impact on the clinical outcome in patients infected with C jejuni. Methods: The levels of CDT expressed by 30 clinical C. jejuni isolates, from patients with campylobacteriosis, were determined using a HeLa cell assay. The presence of the three CDT coding genes, cdtA, cdtB and cdtC was tested in these isolates by PCR. The presence and titres of serum neutralizing antibodies to CDT in the corresponding patient serum was also measured. Clinical data were obtained from these patients using a standardised questionnaire.

Results: PCR examination of the cdt genes revealed that 29 of the strains had the wild type sequence; the remaining strain had major deletions in the CDT genes. Three of the 30 C. jejuni strains had undetectable levels of CDT, 20 strains had low CDT titres while 7 had high titres. CDT neutralizing antibodies were found in all patient sera. The levels of anti-CDT antibodies did not correlate with the levels of IgG, IgM or IgA against whole cell heat-stable C. jejuni antigens.

The results showed a trend in low CDT titre increasing the risk for patients to become febrile. Neither CDT expression nor the presence of CDT neutralizing antibodies in patient serum correlated to clinical outcome. Thus, this study indicated that CDT cannot be used for diagnostic purposes. This study presents 80 patients who had been treated at Clinic for Infectious diseases during the last two years, and who had exhibited the signs of meninges excitation, with or without neurological disorders, but with normal cytological and biochemical findings in cerebrospinal fluid.The most common diagnoses were virosis, bronchopneumonia, pharyngitis, vertiginous syndrome, urinal infections, etc. The majority of the patients were 10-25 years old. Among them, 5% reported previous convulsions, end even 14% informed us of earlier head trauma. Pathological electroencephalographic (EEG) findings were observed in 50% of the male and 63% of the female patients. The EEG disorders occurred mostly between the 5th and the 15th day of the disease. Among the patients with pathological EEG findings, 94% were conscious, 3% somnolent, and 3% soporal. According to the location of EEG changes, the temporal region dominated -27%, and the frontal region was the rarest -8%. Most of them had unspecific EEG disorders, a few exhibited irritative changes, steeper waves or specific changes. In our opinion, earlier convulsions and head traumas in these patients explain way they reacted with meningeal or neurological symptoms to other infection. Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 dissociation step, which resulted in the disruption of the binding between antibody and antigen. The assay protocol was developed and evaluated using well characterised samples collected from HIV infected individuals using a range of serological and molecular assays. An AI of 60% was identified as a threshold below which HIV infection probably occurred within the previous 90 days. Avidity index has the advantage of determining the relative timing of HIV infection using only one sample, which is often the case when performing unlinked epidemiological investigation. Data will be presented showing the application of the assay to the investigation of newly identified HIV infection.

Comparison of Results: 117 patients with bacterial sepsis/severe sepsis were included, 73 (62%) with bacteraemia: 24 with Gram-positive bacteraemia and 49 with Gram-negative bacteraemia. There were no statistically significant differences between either bacteremic and nonbacteremic (p = 0.46) or between Gramnegative and Gram-positive (p = 0.37) bacteremic patients, regarding fever. At cut-off value of 2 ng/ml, PCT discriminate between bacteremic and non-bacteremic septic patients (p = 0.0002) with a positive predictive value of 80% and a sensitivity of 78% for bacteraemia. There are no significant differences for PCT values between Gram positive and Gramnegative bacteremic patients but at cut-off of 10 ng/ml for PCT the positive predictive value was 73% for Gram-negative bacteraemia.

Conclusion: Fever does not have a discriminatory power for bacteraemia in septic patients and the use of PCT assessment could help physicians limit the number of blood cultures to be processed.

Urogenital mycoplasma diagnosis, identification and sensitivity testing to 7 antibiotics I. Gulbinovic, D. Ambrasiene (Vilnius, Kaunas, LT)

U. urealyticum (U.u) and M. hominis (M.h) are species in the family Mycoplasmataceae, which is the smallest bacteria replicating in culture medium and they do not possess peptidoglycan cell walls. These tiny microorganisms can be found commensal in lower genitourinary tracts of sexually active men and women. Moreover, they cause many disorder such as non-gonococcal urithritis, postpartum fever, infertility, and pelvic inflammatory disease. 15 human species have been found to date all belonging to the mollicutae class. They differ from other bacteria in their lack of a cell wall and hence a natural resistance to a-lactams, as well as by the presence of a membrane rich in sterol obtained through their adhesion to eukaryotic cells.

Objective: The aim of present study was to evaluate the occurrence of U.u and M.h in non-gonococcal urethritis and to determine the bacterial resistance to seven antibiotics in order to determine the most suitable treatment strategy.

Methods: A total of 83 patients were enrolled into the study. Urethral samples were taken with a dacron swab placed into urethra 2-3 cm in males, and vaginal samples were taken from the endocervical region in women. Mycofast EvolutioN 3 detects urogenital mycoplasma growth in a liquid milieu. During growth, U.u and M.h metabolise urea and arginine respectively resulting in a colour change of the medium, which contains phenol red indicator, from yellow-orange to red. This colour change is due to liberation of ammonia resulting in an alkaline pH of the medium. Mixed cell granuloma was the most common histologic feature, but suppurative granuloma was the most common histological feature (47.12%) in which AFB could be found, which was statistically significantly different from other types of granuloma. Tuberculoid granuloma was more common in the AFB negative group (10.53%) compared to the AFB positive group (9.37%) but the difference was not statistically significant. In cases that AFB could not be found, the inflammation tended to be located in the upper half of the dermis. Conclusion: AFB can be more frequently detected in suppurative granuloma that might be located in any portion of the dermis.

Multi-drug resistance Mycobacterium tuberculosis in a centre of Tunisia ) ) ) ANA ) ) ) Ferritin (ng/L) 7361 7500 616 of this regional, national and world problem, some intense preventive measures must be applied rigorously.

Determination of Mycobacterium tuberculosis drug resistance in Mazandaran province, Iran -2002 M. Ahanjan, M. Vahedi, M. Nasrollahie (Sari, IR)

Objectives: Tuberculosis (TB) is one of the most important infectious disease and the main cause of mortality in the developing countries (4). Drug resistant leads to failure in treatment which is followed by unaffordable expenses (6). Administrators of Iran to plan necessary educational programmes, and as soon as possible eradicate tuberculosis in Iran. Methods: Sputums from the individuals referring to health centre suspected of having TB were collected in sterile containers, decontaminated by PETROPH method and concentrated, supernatant was removed and collected in container having disinfectant then sediment was inoculated onto the Lowenstein-Jensen medium and incubated at 37 (C for 4 weeks. Smears were prepared from the collected samples, Zeihl-Neelsen staining was performed and looked for the presence of mycobacterium for 10 min growth on the medium 4 weeks after the inoculation it was transferred to the partment of mycobacteriology Culture media were observed for the growth of mycobacterium tuberculosis (TB 

Tuberculosis and diabetes: risks and realities

The vicious combination of tuberculosis (TB) and HIV is now well-established, though not well-controlled. More unnoticed is the meeting between TB and diabetes, predominantly type 2 (DM2,) particularly in low-income countries in fast transition. In India, e.g., about 10% of adults have DM2 and another 10% prestages (impaired glucose-tolerance), while 1% may have active TB and 30% have are at risk of (re-)activating TB. In Mexico, the risk for reactivating TB in patients with DM2 was increased 4-6 times, which is comparable with increased risk for TB among HIV-infected. The World Health Organisation preview that by year 2025 about 350 mio. may have DM2, more than 70% in developing countries. It remains to be seen if TB may double or quadruple in the wake of the DM2 epidemic, as has been the case with TB in HIV. The mechanism(s) behind the increased risk for re-activation of TB in DM is poorly understood. Present knowledge (and lack thereof) will be highlighted and suggestions given for future research and possible measures to control this emerging health problem.

Meningo-encephalo-mielo-polyradiculonevritis with Mycobacterium tuberculosis: case report C. Jianu, R. Iubu, I. Ciutica (Cluj-Napoca, RO)

Objectives: Tuberculosis still represents a public health problem in developing and underdeveloped countries. We report a case of central nervous system tuberculosis.

A 17 year-old female was admitted in our hospital for fever, headache, loss of weight and progressive disability to walk. She presented: fever, later cervical lymphadenopathy and the objective neurological signs were: coma, nuchal rigidity, Kernig signs were positive, lack of abdominal cutaneous reflexes and osteotendinous reflexes, paraplegia, convergent strabismus. We performed: CT brain scan, routine hematological and chemical tests, chest X-ray, lumbar puncture, CSF culture, blood culture.

Results: CT brain scan revealed cerebral edema. Objectives: The increasing incidence of tuberculosis and multidrug resistance (MDR) among Mycobacterium tuberculosis strains remains one of the major public health problems in Poland. The geographical position of Lublin means that potentially MDR strains from former Soviet Union countries are more likely to be present. Rapid detection of MDR can optimize the efficacy of antituberculosis therapy and control their transmission. The aim of the study was to compare current methods of drug susceptibility testing with two molecular assays. Methods: The Inno-Lipa Rif. TB was applied to detect mutations in rpoB gene conferring rifampicin resistance and a noncommercial low-density macroarray (MDRTB screening array) detecting mutations in rpoB and kat G, inh A genes responsible for rifampicin and isoniazid resistance, respectively. Differentiation of M. tuberculosis complex species was confirmed using biochemical analyses, niacin accumulation test and spoligotyping based on polymorphism of the chromosomal direct repeats (DR) locus, which contains a variable number of short DR interspersed with non-repetitive spacers.

Results: 18 MDR strains cultured from patients with pulmonary and extrapulmonary tuberculosis in the laboratory of Public Hospital were subjected to susceptibility testing for rifampicin and isoniazid by the resistance ratio method on L-J medium. All these strains were analysed by the Inno-Lipa Rif, MDRTB screening array and spoligotyping. Phenotypic testing established that 17 of 18 isolates were rifampicin and isoniazid resistant using the resistance ratio method -Ôthe gold standardÕ to compare with. Objectives: To evaluate the performance of BacT/ALERT 3D (bioMerieux, France) for isolating of mycobacteria in comparison with the Lö wenstein-Jensen medium. Additionally, a series of studies were conducted to detect the sensitivity of p-nitrobenzoic acid test for differentiation of Mycobacterium tuberculosis complex with other mycobacteria at Social Security Institute Sureyyapasa Chest Disease Hospital, Istanbul. Methods: During our investigation, a series of different samples such as sputum, bronchial aspiration, pleural fluid, bronchoalveolar lavage, gastric lavage, urine, periton fluid and lenfadenopathy drenage recovered from patients in the three major services, were treated with N-acetyl-L-cysteine-3% NaOH decontamination and concentration procedure. Each sample was then cultivated in BacT/Alert 3D MP bottle and Lö wenstein-Jensen medium in 37°C. Acid-fast staining was employed for smear preparation. Time and presence of growth on BacT/Alert 3D MP bottle was monitored and recorded. Additionally, we have evaluated efficiency of BacT/ Alert MP medium in rapid differentiation of mycobacteria by adding PNB.

Results: Among the smear-positive and smear-negative specimens, more Mycobacteria grew on BacT/ALERT MP then L-J media. In smear-positive samples, BacT/ALERT 3D system showed a few days earlier then in smear negative samples. Contamination ratios for BacT/Alert 3D and L-J were 3 and 4%, respectively. The total mean time of detection of BacT/ ALERT MP bottles were approximately 19 days shorter than Lö wenstein-Jensen media. It has been determined that all bottles with Mycobacterium tuberculosis complex showed no growth or late growth compared to bottles containing p-nitrobenzoic acid. But other mycobacteria bottles showed same type of growth as the bottles containing p-nitrobenzoic acid. Mean time of p-nitrobenzoic acid test for other mycobacteria and Mycobacterium tuberculosis complex have not exceed more than 5 days.

Conclusions: It has been showed that the performing of p-nitrobenzoic acid test in BacT/ALERT MP bottles is sensitive and rapid. Thus total testing time of mycobacteria at the system provides the ability to meet the 21 days target for detection and identification of M. tuberculosis complex as recommended by the Centers for Disease Control and Prevention, Atlanta, GA, USA.

Early diagnosis of lung tuberculosis by automatic system probetec ETTH (Becton-Dickinson) Results: A total of 61 (31.3%) samples were positives for MTC DNA, of these samples 49 (80.3%) were BC positives and 12 (19.7%) were negatives. In those 3 samples (1.5%) with BC positive and MTC DNA negative, atypical mycobacteria were presented in 2 cases and in the other one a culture contamination eluded its identification. MTC DNA positive and BC negative were in 12 samples (6.1%), in these MTC only could be demonstrated in 4 (33.3%), in the others the half were from patients with LTB therapy started.

Conclusions: SDA method is quickly, efficient and easy to the early diagnosis of LTB (4-6 h) overall in cases with BC positives. We have achieved a sensibility of 94.2% compared with BC and a specificity of 91.6%. We consider that this technique cannot be used by LTB therapy monitorization. Multidrug-resistant tuberculosis, a disease caused by Mycobacterium tuberculosis strains that are resistant at least to rifampin and isoniazid, entails extended treatment, expensive and toxic regimens, and higher rates of treatment failure and death. To document the presence of multidrug-resistant tuberculosis (MDR-TB) strains in patients from Turkey on Black Sea region between January 2000 and October 2004. We retrospectively analysed the outcomes in 249 patients treated and untreated , and tests of primer and seconder resistance to isoniazid, rifampicin, streptomycin, ethambutol and multi drug resistance on their specimens were performed using the Bactec TB-460 system. The most commonly encountered culture-positive specimens were sputum (67%) and BAL fluids ( Objectives: Tuberculous brain abscess (TBA) is a rare clinical entity, even in endemic areas. These lesions may occur during the treatment of tuberculous meningitis. Drug resistances were not well defined in Central Nervous System tuberculosis.

Case report: A 19 years-old, immunocompetent, young man was admitted with diplopia, nausea, vomiting and a change in mental status. The patient had a history of tuberculous meningitis diagnosed before six months in another hospital and given anti-tuberculosis treatment with isoniazid (INH), rifampicin (RF), pyrazinamide (PRZ) and ethambutol (ETB) for two months and followed by two drugs treatment with INH and RF for four months. Computed tomography (CT) scan of the brain showed a non-communicating hydrocephalus. The patient was operated for hydrocephalus and placed a ventriculo-peritoneal shunt. Two mounts later, the patient was hospitalized again for fever, dysfagia and left hemiparesis. Cranial CT scan was within normal; however magnetic resonance imaging (MRI) examination revealed irregular multiloculer peripheral contrast enhancement lesion with hypointense necrotic centres within the left cerebellar hemisphere extended to the upper posterior cervical subarachnoid space through left medullacerebellar fissure. The abscess was drained by surgical operation. Acid resistant bacillus (ARB) was demonstrated in the abscess material by Zeihl-Neelsen stain and polymerase chain reaction (PCR). Isoniazid resistant Mycobacterium tuberculosis was grew on Lowenstein-Jensen culture medium. One month after the operation quadryparezi was developed. Cervical MRI was revealed cervico-thoracic syringomyelitic cavity. The treatment was given with four drugs (INH, RF, PRZ, and ETB) for 10 months and it was continued with three drugs until 24 months.

The patients with tuberculous meningitis should be observed closely for complications like brain abscess and all isolates of M. tuberculosis must be tested for drug resistance.

Pneumocystis jiroveci in patients with pulmonary infections L. Nimri, J. Amer (Irbid, JOR)

Objectives: In patients with acute or chronic respiratory problems mainly tuberculosis (TB) suspected cases, there is a concern that other opportunistic infections apart from TB, such as Pneumocystis jiroveci, may be missed owing to lack of diagnostic facilities. The aim of this study was to investigate the extent of P. jiroveci pneumonia in patients with respiratory tract infections especially in TB patients registered for smear-negative. Methods: Three hundred specimens, included bronchoalveolar lavage, and sputum collected from patients having different pulmonary infections were tested by nested PCR for P. jiroveci and TB. Results: P. jiroveci was detected in 15 (5%) patients, 7 (46.7%) were TB negative by both acid fast-smear and PCR, the other 8 (53.3%) had dual infections with TB. These patients included old patients, alcoholic, heavy smokers, and post primary TB patients. Results suggest that these might be risk factors for Pneumocystis pneumonia (PCP). Patients with PCP had somehow different clinical presentation and radiographic appearances from patients with TB. The PCP infections might be overlooked due to concurrent infections with TB, which makes the diagnosis difficult.

Conclusions: The use of sensitive diagnostic methods for the diagnosis of PCP in patients with respiratory tract infections who are not responsive to treatment is recommended.

Susceptibility testing of pathogenic fungi with fluconazole and tea (Camellia sinensis) on Candida albicans PTCC-5027 Z. Nasrolahi (Tehran, IR)

Tea (Camellia sinensis) has recently been shown to inhibit growth of a range of bacteria and microfungi. In my study Candida albicans standard strain PTCC-5027 were tested for in-vitro susceptibility to fluconazole and tea containing whole extract of Lahijan fresh tea leaves and black tea, Chinese green tea and tea polyphenols and caffeine. A rapid reversed phase HPLC method for the determination of polyphenols, using a binary gradient system, was developed. At the present research, which is aiming at determining the minimum inhibitory concentration of tea extract and tea polyphenols and caffeine at concentration (6.26-200 mg/ml) on (500, 1000, 2000 cells Candida albicans PTCC-5027) at two period 24 and 48 h. The macrodilution broth test technique was performed to do this. Fluconazole was used for comparing in this study, and we found resistance of that strain with fluconazole and MICs > 64 lg/ml. The 100% minimum inhibitory concentrations (MFC) calculated in our study are evidence of antifungal activity of polyphenols against fluconazole-resistance Candida albicans PTCC-5027. The obtained results confirmed that caffeine in rather than polyphenols produced better treatment outcomes. The obtained MFC for polyphenols in comparison to the whole extract of tea has better inhibitory effect at 48 h on Candida albicans PTCC-5027.

Aspergillosis of the maxilla sinus in patients from maxillofacial surgery clinic in Cracow, Poland, between 1998 and Budak, A. Dzierwa, B. Bogusz, J. Zapala, P. Nowak (Cracow, PL)

Objectives: The aim of the paper was to analyze the occurrence of the aspergillosis of maxilla sinus among patients hospitalized in Maxillofacial Surgery Clinic connected with the estimation of the results of laboratory-clinical diagnostics applied and together with therapeutical procedure.

Methods: The subject of the analysis was the group of 398 patients with chronic inflammation of the maxilla sinus hospitalized in the clinic between 1998 and 2004. The diagnosis of aspergillosis was performed on the basis of interview, object, subject studies, radiological studies, mid-operation estimation of the state of mucous membrane and inside of sinus, and on the basis histological and mycological examinations. Aspirates taken during operation from maxilla sinus were subject to mycological investigation. Diagnostics included: direct microscopic examination, culture on Sabouraud agar, microculture and estimation of MIC for itraconazole and voriconazole using E-tests.

Results: Aspergillosis of maxilla sinus was diagnosed in 12 patients (5 males, 7 females) in age from 19 to 74, respectively in years: 2000: 2, 2001: 1, 2002: 2, 2003: 5 and 2004 : 2 cases. In mycological examinations in 5 patients the presence of septate mycelium was proved in direct preparations and in one case the presence of Aspergillus conidiophores was also stated. In 3 persons positive result from preparation was proved by culture. Moulds from genus Aspergillus was identified. In 2 patients isolates were recognized as Aspergillus niveus and A. candidus. In histological examination in 9 patients the presence of foreign body was also stated in lumen of sinus and numerous septate mycelium was also identified in preparations. All patients were treated with combined surgical and antifungal therapy using itraconazole and in one patient ambisone. Conclusions: Applied therapeutical procedure caused significant improvement of local and general condition of patients, which was confirmed by the results of mycological investigations and by the interview which took place along time after surgical operation. In one patient invasive form of aspergillosis was observed together with the expanding inflammation process to eye-socket and to lower part of front skull. Objectives: Amphotericin B deoxycholate (AmBd) has been a standard therapy for most systemic mycoses. It is associated with significant adverse events (AEs) including nephrotoxicity (NT). Objectives were to describe AmBd therapy, AmBd related AEs, and clinical outcomes. Methods: Retrospective chart review was undertaken in an academic centre in Geneva, Switzerland, on patients with IFI receiving AmBd as initial therapy. Data abstracted included: AmBd mean daily doses (MDD), AEs, clinical/microbiologic response to antifungal treatment (AFT), and mortality. NT: 50% increase in baseline serum creatinine (SCr) or if baseline SCr > upper normal limit, an absolute increase of 1 mg/dL. Success was defined as complete or partial clinical response to the At that time he was admitted to a hospital outside Croatia and treated for mixed bacterial (Streptococcus sanguis) and fungal (Candida albicans) sepsis with tricuspid valve endocarditis. His hospitalization was complicated by pulmonary embolism, femoral thrombosis and bilateral pneumonia that required bilateral thoracic drainage. His Candida albicans infection was treated with liposomal amphotericin B for 3 weeks followed by oral fluconazole. He was discharged in December 2003 in a stable condition and given fluconazole (400 mg/day) and methadone maintenance therapy. Case presentation: The patient was admitted to our hospital in March 2004 because two blood cultures taken in the outpatient setting grew Candida albicans despite continuous fluconazole therapy. Echocardiography was performed and a vegetation 2 cm in diameter was found on the tricuspid valve, and there was also a thrombotic mass 0.5 cm in diameter in the right ventricle. A 2-week course of liposomal amphotericin B with intravenous fluconazole (400 mg/day) was given, followed by oral fluconazole (400 mg/day). The patient became afebrile, however, a blood culture taken in May 2004 grew Candida albicans again. On June 2nd surgery was performed with excision of the vegetations on the tricuspid valve and in the right ventricle. One the day prior to surgery treatment with intravenous caspofungin (70 mg on day 1 followed by 50 mg/ day) was started. Candida albicans grew from the cultured vegetations, it was susceptible to fluconazole and amphotericin B. Intravenous caspofungin was given for 4 weeks followed by oral fluconazole (400 mg/day). In the following 4 months no recrudescence of endocarditis was observed. Antiretroviral treatment was started in April 2004 with a combination of stavudine, lamivudine and lopinavir/ritonavir. It was given throughout the course of illness and was well tolerated. Conclusion: Initial caspofungin treatment followed by fluconazole maintenance therapy, in addition to vegetectomy, was successful in the treatment of Candida albicans right-sided endocarditis in our HIV infected patient.

Survey of the prevalence and identification of dermatophytosis in Northern Iran, 2003 Iran, -2004 J. Hashemi, A. Nasrollahi Omran (Tehran, Tonekabon, IR)

Objective: For 15 months in the period from June 2003 to September 2004, we experimented 400 patient suspected to dermatophytosis refer to clinics medical mycology to have itching and irritation. Materials and methods: Skin samples were taken by scraping from patients and collected. Diagnosis was confirmed by direct microscopy carried out with a 20% KOH preparation and culture were performed in Petri dishes on mycobiotic agar and dermatophyte medium (DTM), and dermatophyte diagnosis tests for taxanomic identification of species. Thus after to complete questionnaire, we analysis prevalence of dermatophytosis.

Results: Our results showed that: Totally, that 210 cases of them suffered from dermatophytosis, that disease confirmed by microscopic examination and culture positive. %70 (147 case) of this patients suffered from disease were male and rest were female 30% (63 case). Age groups between 10 and 50 were effect by this disease, with a lightly higher incidence in the age group was 20-30 (57%). Seventy per cent (147 case) of patients living in the cities rest 30% (63 case) were villager. Majority of the patients were driver 30% (63 case), rest were student 20% (42 case), officer 20% (42 case), collar worker 20% (42 case) and 10% (21 case) farmer. The frequency of clinical types according to the anatomic site involvement of dermatophytosis was groin, thus tinea cruris 50% (105) was the predominant clinical form of all ring worm infection see, rest were 20% (42 case) of cases were tinea manum, 17.6% (37case) tinea pedis, 4.76% (10 case) tinea capitis. 4.76% (10 case) tinea corporis, 1.9% (4 case) tinea unguium. Ninety five per cent (2 case) tinea barbae. Eighty per cent of tinea capitis was ectothrix rest were 5% endothrix and 15% tinea favosa. Epidermophyton floccosum was most common etiological agent 28.5% (60 case) rest encountered dermatophytosis including: T. rubrum 23.8% (50 case); T. violaceum 7.14% (15 case); T. mentagrophytes 14.28% (30 case); T. verrcosum 4.76% (10 case); T. schoenleinii 11.9% (25 case); T. sonsudens 4.76% (10 case); M. canis 476% (10 case). We found that anthropophilic infections 85.72% (180 case) appeared to be six time frequent than zoophilic ones 14.28% (30 case) Conclusion: Statistical evaluation of obtained data indicated the decreased trends towards frequency of tinea capititis and the growing trends toward tinea cruris. This study highlights a common problem in many areas of the northern Iran and suggests that further measures regarding public health and specially personal hygiene must be undertaken in order to reduced the risk of dermatophytosis.

Colonisation of oral cavity by Candida species Objectives: The aims of the study were to assess the colonization of oral cavity in otherwise healthy individuals with oral symptoms (dry mouth and burning mouth syndrome) by Candida species and to determine their susceptibility to antimycotic drug nystatin. Nystatin is frequently used for topical prophylaxis or treatment of oral candidiasis.

Methods: This study examined the oral yeast colonization in 104 participants. Samples were collected with tongue swabs and cultured on Sabouraud's dextrose agar at 37°C for 48 h. All isolates were identified by germ tube production, chlamydospore development on cornmeal agar with 0.5% Tween 80 (Difco, Detroit, USA) and ID 32 Candida identification kit (bio-Merieux, Marcy-l'Etoile, France). The susceptibility profile was evaluated by disk diffusion method for nystatin (NeoSensitabs, Taalstrup, Denmark) on Mueller-Hinton agar.

Results: At the time of sampling, 30 (28, 84%) individuals were positive on colonization by Candida species. 27 (90%) of isolates corresponded to Candida albicans while two isolates (6.6%) were identified as C. kefyr (C. pseudotropicalis) and one (3, 3%) as C. glabrata. All the isolates were susceptible to nystatin. Conclusion: Candida albicans was the main Candida species that colonized oral cavities while non-C. albicans species were rarely detected. Since all isolates were susceptible, nystatin can be safely used for prophylaxis of oral candidiasis in patients with oral symptoms such as dry mouth or burning mouth syndrome.

Confirmation of C. dubliniensis from other Candida spp. Objectives: C. dubliniensis (CD) is often identified as C. albicans (CA) specially in routine mycological practice because two species shares characteristics, they are Ôgerm-tubeÕ positive and produce chlamydospores. The aim of this study was to distinguish CD from other Candida spp. in immunocompetent and immunodeficient patients clinical specimens. Methods: All Candida spp. isolates were tested using algorithmtolerance at 45°C, osmotic resistance in broth containing 6.5% of NaCl and phenotypic features on CHROM agar and Pal agar. For quality control we used C. albicans ATCC9028 and C. dubliniensis CBS7988. Results: One hundred and twenty three strains of Candida spp. isolated from genitourinary tract of immunocompetent patients were tested for CD and was no such strain confirm. In 93 Candida spp. from oral cavity in haematological patients and 10 swabs from HIV positive persons we confirm only one strain as CD. CD growth on Sabouraud-agar at 37°C and do not at 45°C does not growth in broth with 6.5 % NaCl, on Pal agar colonies have characteristic pseudo-hyphal roots (with chlamydospores under 400·). On CHROMagar CD have dark blue-green colour distinguishable from light-blue CA colonies. Conclusions: Using algorythm (ÔgermtubesÕ) and chlamydospore formation together with two methods: growth at 45°C and cultivation on Pal agar present easy methods for distinguish C. dubliniensis from C. albicans and other Candida spp. in clinical laboratory specimens.

Candidosis due to Candida guilliermondii: an analysis of eleven cases Objectives: In the attempt to separate colonization from infection, we reviewed the clinical records of patients who had Candida guilliermondii identified on clinical specimens. Methods: Retrospective study of all positive cultures for C. guilliermondii obtained during October 1997 to August 2003 at Santa Casa Complexo Hospitalar, Brazil. The records of the Mycology Laboratory were reviewed attempting to identify positive cultures to C. guilliermondii. Clinical records of these patients were also reviewed. Candidemia was diagnosed if a peripheral positive blood culture was temporally related to signs and symptoms. Histopathologic or cytopathologic examination showing C. guilliermondii from specimens of needle aspiration or biopsy, excluding mucous membranes, were considered evidence of invasive infection, as well as positive culture result on sample obtained by sterile procedure from normally sterile site. Results: Eleven patients were included in this study. Specimens corresponded to blood (n = 5), ascitis fluid (n = 2), pericardial fluid (n = 1), urine (n = 1), colon biopsy (n = 1), and esophagus mucosa (n = 1). Patients were mainly male (54%), and age ranged from 19 days to 73 years. Six patients were immunosuppressed (3 transplant recipient, 2 AIDS, and 1 cancer), and 4 had been exposed to invasive procedure (2 peritoneal dialysis, and 2 abdominal surgery). For the 5 patients with candidemia, there was a favorable response with amphotericin B treatment.

Only one case, an immunocompetent patient, received no antifungal therapy. Two cases were presented of C. guilliermondii peritonitis, associated to dialysis catheter; both had favorable evolution after antifungal therapy and catheter removal.

Conclusions: In this series of 11 patients, we demonstrated that, although C. guilliermondii can be associated with invasive disease, its presence may represent colonization or even infection with low pathogenicity, mainly in immunocompetent patients. There was a frequent association between C. guilliermondii and catheter use (intravenous, peritoneal or urinary), which is common to Candida species infections. Aseptic technique should be rigorously performed during the process of blood sampling, to avoid further mistakes in the interpretation of the isolation of C. guilliermondii from clinical specimens.

Importance of the coexistence of Candida fungi and bacteria Helicobacter pylori Objectives: Except of well known role of Helicobacter pylori (Hp) in the pathogenesis of ulcer disease (UD) and chronic gastritis (CG), the role of the presence of Candida (C.) fungi in the stomach is now evaluated. The problem of the coexistence of Candida and Helicobacter pylori in the gastric mucosa and their influence on ulcer healing and the course of chronic gastritis has not been explained. The aim of study was to (1) evaluate the frequency of C. in patients with gastric ulcer (GU), duodenal ulcer (DU) and CG; (2) estimate the frequency of Hp in GU, DU and CG; (3) evaluate the frequency of coexistence of C. and Hp in above patients; (4) identify the C. species, isolated from patients with GU, DU and CG; (6) evaluate the influence of Hp eradication on the frequency of the presence of C. in the stomach. Methods: We examined the group of 63 patients, including14 patients after Hp eradication therapy, aged from 18 to 72 years, with dyspeptic and ulcer symptoms. Clinical investigation included: clinical history, gastroscopy with biopsies for urease test, histopathology, mycology and evaluation of the Hp presence. Also brush smear from changed gastric mucosa and aspirate of gastric juice were taken for mycological investigation. Bacteriological investigation included: (a) Hp culture in the incubator with CO 2 flow, (b) identification of Hp species by evaluation of urease, catalase and oxydase activity. Mycological investigation of examined material was evaluated on the basis of qualitative and quantitative investigation. In the identification of isolated fungi: Albicans ID2, ID 32C ATB System (bio Merieux) was used.

Results: (1) Presence of C. in the stomach was found in 47% and 57% patients before and after Hp eradication therapy respectively. (2) We did not find C. and Hp in 53% and 29% in not eradicated and eradicated patients respectively. (3) Coexistence of C. and Hp in the stomach was found in 10% of cases. (4) C. albicans was the most frequently isolated species. Conclusions: (1) C. and Hp coexistence was found in 10% of cases.

(2) Hp eradication therapy containing proton pump inhibitor and antibiotics may increase the frequency of fungal colonization of stomach mucosa.

Molecular characterisation of GTP-binding protein gene in dermatophyte pathogen Trichophyton rubrum S. Rezaie, F. Nourbakhsh, F. Fallahyan (Tehran, IR)

Trichophyton rubrum (T. rubrum) is an anthropophilic dermatophyte. Infections caused by the fungus are mostly confined to the keratinized epithelial layer. T. rubrum is the most common cause agents of dermatophytosis in human skin and nail tissue. Some properties of T. rubrum have been investigated in molecular level, however, no information is available regarding the GTP-Binding protein in this dermatophyte. GTP-Binding proteins regulate a variety of processes including sensual perception, protein synthesis, various transport processes, and cell differentiation. In the present study, we try to characterize the GTP-Binding protein gene in this dermatophyte pathogen.

T. rubrum was obtained from patients with dermatophytosis and cultured in appropriate conditions. Nucleic acids (DNA and RNA) were isolated from obtained mycelial mass by standard methods. Pairs of 21 nt primers were designed from highly conserved regions of the GTP-binding protein genes in other eukaryotic cells. Mentioned primers were utilized in PCR by using isolated genomic DNA as well as cDNA template of T. rubrum. Predicted molecules have been amplified and were sequenced. By the time 350 nucleotides of this gene are sequenced. The characterization of this PCR fragments which revealed significant homology with other GTP-binding protein in GenBank (NCBI, NIH, USA) is still under investigation. Trichophyton rubrum (T. rubrum) is one of the dermatophyte fungi which invade the skin of human. Several properties of this fungus have been investigated so far. However a few studies were carried out in the field of molecular biology of this fungus. In the present study we tried to identify the vacuolar ATPase proteins (V-ATPase) in this fungus. Pairs of 21 nt primers were designed from highly conserved regions of the same gene in other fungi. Mentioned primers were utilized in PCR by using isolated genomic DNA as well as cDNA template of T. rubrum and the PCR fragments were then sequenced. By the time, 568 nucleotides have been sequenced from this new gene which encodes a polypeptide with 119 amino acids. Nucleotide sequence comparison in gene data banks (NCBI, NIH) for both DNA and its deduced amino acid sequence revealed significant homology with V-ATPase genes and proteins from other eukaryotic cells. The molecular characterization of this gene as well as definition of its possible role in the physiological function of T. rubrum are still under investigation. The new gene has been submitted to the international GenBank (NCBI, NIH, USA).

Aspergillus arthritis in an immunocompetent patient A. Azap, Ö . Demir, A. Kaya, D. Yagci, E. Tekeli, H. Gü riz, N. Numanoglu (Ankara, TR)

Case: Aspergillus is an opportunistic pathogen that causes severe clinical diseases in immunosuppressive patients. It is widely distributed in nature and rarely causes disease in immunocompetent patients. A 69 year-old immunocompetent female patient was admitted to our hospital one and a half month after implantation of bilateral knee prothesis. She had no other underlying disease. Her complaints were fever, cough, hemoptysis, pleuritic chest pain and dyspnea on the 5th week after operation. She was hospitalized with the suspicion of pulmoner thromboembolism and anticoagulant therapy was started. The purulan drainage from both knees was noticed during the follow up. Polymorphonuclear leukocytes and Grampositive cocci were seen in the microscopic examination of the joint fluid. Teicoplanin treatment was begun. She improved, fever resolved but necrotic areas appeared during teicoplanin treatment. The pathological examination of the necrotic material demonstrated fungal hyphae and the culture grew Aspergillus niger. Amphotericin B was started along with debridman and the patient improved dramatically. After 42 days of amphotericin B therapy, both knees and the patient were well without prothesis replacement. She was still in good health three months after discharge. Conclusion: Prosthetic joint infections are troublesome complications of orthopedic surgery. Usually Gram-positive microorganisms cause such infections but in descending order Gram-negative bacilli or Candida spp. may be the causative agents. Aspergillus arthritis is a rare clinical entity especially in immunocompetent patients. Other interesting side of this case is the rescument of the prothesis with medical therapy and debridman only.

Fungaemia by yeasts in a Portuguese university hospital: epidemiology and susceptibility profile over a 2-year period C. Pina-Vaz, A. Gonçalves-Rodrigues (Porto, P)

Fungaemia is reported as the 4th cause of septicemia in the international literature, with inherent serious morbidity and significant mortality. No local epidemiological data on this problem are yet available.

Objectives: To define the prevalence of Fungaemia due to yeasts, respective risk factors, reservoirs, distribution by species and its susceptibility antifungals pattern. Methods: All the yeasts isolated from blood cultures during 24 months (2002) (2003) (2004) at an university hospital of Porto were collected. Data like demographical aspects, hospital department, presence of central venous catheter or other indwelling medical devices, concomitant diseases, as well as antimicrobial therapy were collected. Additionally, concomitant isolation of yeasts from distinct body sites was also registered. The nosocomial nature of such infections was investigated. Following biochemical characterization of isolates, performed by Vitek system (BioMérieux), the susceptibility pattern to antifungals like amphotericin B, fluconazole, itraconazole, voriconazole and caspofungin, was determined according to NCCLS protocol M27-A2.

Results: A total of 160 blood yeasts strains were identified, representing 0.3% of all positive blood cultures; 31% from Internal Medicine, 24% from Surgical Departments, 18% from Hematology/Oncology and 17% from Infectious Diseases Department. C. albicans and C. parapsilosis were isolated in similar percentage, 33.5%; 12.8% were C. tropicalis; 7.7% C. glabrata; 6.9% Cryptococcus neoformans; 2.5% C. guillermondii and 1.25% C. lusitanea. All the strains were susceptible to amphotericin B except C. lusitanea; 20% of isolates were resistant (R) to fluconazole, 7% being susceptible dose-dependent (S-DD); 26% were R to voriconazole (MIC > 1 lg/ml); 22% R to itraconazole while 30% S-DD; 41% were R to caspofungin (MIC > 1 lg/ml). Conclusion: Fungaemia due to yeasts represents is an important concern among us, being non-albicans species of Candida prevalent comparing with other international studies. Due to this fact, resistance to antifungals was found in a significant extent.

C. krusei showed promoted resistance to oxidative stress S. Costa-de-Oliveira, C. Pina-Vaz, A. Gonçalves-Rodrigues, P. Ludovico (Porto, Braga, P)

Candida is of great concern as such isolates are more resistant to antifungals. C. krusei is a paradigma example, whose pathogenicity is not yet full understood, being often negative for mechanisms like germ tube formation or production of enzymes like coagulase (1). Recent studies have implicated a role for adaptative responses to oxidants in pathogenesis. The reactive oxygen species (ROS) are produced mainly by phagocytic cells of the host immune system, its microbial effect leading to the destruction of potential pathogens.

Objectives: To study the response of C. krusei to some oxidative stress agents. Methods: Blastoconidea of 10 clinical strains of C. krusei and 10 strains of C. albicans were stained with 50 lg/ml of MitoTracker Red CM-H2XRos (Molecular probes) during 15 min and exposed to oxidant stress agents: 0.4 mM of H 2 O 2 , 0.5 mM of menadione and 3 lM of plumbagine, all from Sigma, during different periods of time (15, 30 and 60 min). After treatment the cells were analysed in a flow cytometer (Beckman Coulter XL-MCL). MitoTracker Red CM-H2XRos does not emit fluorescence until entering an active respiratory cell, where is oxidized by agents of ROS. Strains affected by those agents, ROS, will show an increase in intensity of fluorescence.

Results: All C. albicans showed an increase of the intensity of fluorescence after being exposed to oxidative stress agents, which increased with the time of incubation. Conversely, minor variations of the intensity of fluorescence were seen in all C. krusei strains tested. Conclusions: C. krusei are relatively protected to agents of ROS compared to C. albicans strains. Further work is need to clarify its importance and role regarding pathogenicity (1) Objectives: To determine species of Candida fungi inducing respiratory tract infections in pulmonary tuberculosis (TB) patients and analyze their resistance to antimycotics. Methods: Culture (inoculation onto Sabouraud medium with chloramphenicol) and microscopy of bronchoalveolar lavage, sputum (we estimated quantitative concentration of Candida spp. in specimens), samples from lung cavities and pleural cavity (aspirates, biopsies), lung tissue samples (resection); identification of isolated strains (morphology on corn-meal agar and CandiSelect medium, ÔAuxacolorÕ test-system, Bio-Rad); antimycotic resistance determination (using microdilution method validated with NCCLS M27-A reference method -ÔFungitestÕ test-system, Bio-Rad). Results: During 2002-2004 we studied 502 patients with different forms of pulmonary TB. Inoculation of clinical samples revealed strains of Candida spp. in 163 patients (32.5%). In 89.2% from this group culture results were confirmed by positive microscopy of clinical samples. We detected 8 species of Candida. Detection rate of C. albicans strains was 64%, C. glabrata -11%, C. krusei -9%, C. tropicalis -8%, C. parapsilosis -4%, C. kefyr -2%, C. guilliermondii -1%, C. lusitaniae -1%. Susceptibility to antimycotics (fluconazole, itraconazole, amphotericin B, ketoconazole, miconazole, 5-fluorocytosine) was tested in 328 Candida strains (211 strains of C. albicans, 41 -C. glabrata, 27 -C. tropicalis, 24 -C. krusei, 16 -C. parapsilosis, 5 -C. kefyr, 3 -C. guilliermondii, 1 -C. lusitaniae). Resistance to fluconazole was detected in 5% of all studied strains (in 2% strains of C. albicans, 15% -C. glabrata, 21 % -C. krusei and 4% -C. tropicalis). Resistance to itraconazole was in 7% strains (in 6% strains of C. albicans, 17% -C. glabrata and 15% -C. tropicalis).

We also found strains resistant to miconazole (1% strains), ketoconazole (3% strains) and 5-fluorocytosine (2% strains). Strain of C. lusitaniae was resistant to amphotericin B.

Conclusions: Pulmonary TB patients administered long-term adequate course of chemotherapy (4-7 antituberculosis drugs) compose a risk group for candidosis. Considering the experimental data on Candida clinical strains resistance, we deem that before administration of specific treatment for pulmonary candidosis with fluconazole and itraconazole it is advisable to identify the strain and determine its susceptibility to antimycotics.

Candida species isolated from blood and other sterile fluids: distribution and susceptibility Objective: During the last decades yeast infections have emerged as important causes of nosocomial infections. As Candida infections are associated with significant morbidity and mortality, monitoring of Candida species distribution among significant isolates and their resistance is required. Material and methods: All consecutive strains of Candida spp. isolated from blood and other normally sterile fluids cultures at the General Hospital of Athens ÔG. GennimatasÕ during a 5.5-year period (1999) (2000) (2001) (2002) (2003) (2004) were examined. Isolates were speciated by the Vitek II system (BioMerieux, France). Susceptibility testing to amphotericin B, fluconazole, flucytosine, itraconazole and voriconazole was performed by the E-test (AB Biodisk, Sweden); result interpretation was according to NCCLS.

Results: A total of 81 Candida spp. strains were isolated from a respective number of patients hospitalized in Surgery (n = 27, 33.5%), Internal Medicine (n = 39, 48%) or ICU (n = 15, 18.5%); 18 persons (22%) had received fluconazole prophylaxis previously. Sixty five (80%) strains originated from blood and 16 (20%) from other sterile fluid cultures. Nine Candida species were identified. C. albicans represented the most common (n = 32, 39.5%) followed by C. parapsilosis (n = 19, 23.5%), C. glabrata (n = 14, 17.3%) and C. tropicalis (n = 8, 9.9%). However, C. albicans isolation rates decreased steadily from 1999 to 2004 (from 58.3% to 22.2%), while C. glabrata isolation rates increased (from 8.3% to 33.3%). All strains were susceptible to amphotericin B (MIC, <1 mg/l) and a single C. krusei strain (1.2%) was flucytosine resistant (MIC, 3 32 mg/l). Resistance to fluconazole (MIC, 3 64 mg/l) and itraconazole (MIC, 3 1mg/l) was observed in 15 (18.5%) and 24 (29.6%) strains, respectively. C. glabrata and C. krusei were the species more often resistant to the azoles (9 out of 14 and 2 out of 2 strains, 64.3% and 100%, respectively), while C. albicans was the least resistant (1 out of 32 strains, 3.1%). Resistance to voriconazole (MIC, >1 mg/l) was detected among 6 (7.4%) isolates, 3 strains of C. glabrata and 2 strains of C. parapsilosis.

Conclusion: Similarly to data from other countries, our results from Greece show increasing isolation rates of species other than C. albicans in blood and other sterile fluid cultures. Increased frequency of C. glabrata strains resistant to conventional azoles was detected, but voriconazole demonstrated good in vitro activity against Candida species. Results: The ratio of isolates to patients with B. capitatus during the study period was: 1990-1996 (5/5), 1997-2003 (75/39) . Among the clinical isolates of BC, 23 were collected from deep sites (9 blood, 7 lower respiratory tract, 5 sterile fluid, 1 biopsy and 1 abscess) and 67 were recovered from mucosal sites with no systemic involvement. The overall MIC90 (mg/l) of AMB, FC, FZ, IZ, KZ, VZ and AL against 45 available BC were: 0.5, 1, 32, 1, 1, 0.5 and 8. There were no differences in susceptibility between isolates from deep and mucosal sites.

Conclusions: A clear increase in the rate of isolation of B. capitatus occurred during the second period of the study in our institution. Although there are no established breakpoints to define antifungal susceptibility to BC, voriconazole and amphotericin B appeared to be the most active antifungal agents. Our data shows the reduced susceptibility of BC to flucytosine, fluconazole, itraconazole, ketoconazole and anidulafungin.

Evaluation of Candiselect Ò 4 a chromogenic medium for yeasts differentiation U. Nawrot, K. Wlodarczyk, J. Skala, A.P.M. Przondo-Mordarska, N. Nolard (Wroclaw, PL; Brussels, B)

Objectives: Candiselect Ò 4 (BioRad) is a chromogenic medium recommended by manufacturer for selective isolation of yeasts, direct identification of Candida albicans and presumptive Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 identification of C. glabrata, C. tropicalis and C. krusei. The identification is based on the species specific color and colony morphology, namely: pink-purple for C. albicans, dark turquoise and smooth for C. tropicalis, dark turquoise and rough for C. krusei and pale turquoise for C. glabrata. In this study we evaluated the performance of Candiselect Ò 4 for the identification of clinical yeasts isolates. Methods: A total number of 204 strains included 57 C. albicans, 43 C. glabrata, 18 C. krusei, 13 C. tropicalis, 23 C. parapsilosis, 7 C. inconspicua, 6 C. kefyr, 6 C. guilliermondi, 4 C. dubliniensis, 2 C. rugosa, 1 C. norvegensis, 1 C. lusitaniae, 1 C. lipolytica, 19 S. cerevisiae, 2 C. neoformans, and 1 P. anomala. 13 strains were obtained from BCCM IHEM Collection and 191 were a clinical isolates collected in Department of Microbiology Medical University of Wroclaw and identified previously using standard methods (e.g. ID-32C tests, BioMérieux) and on the base of rDNA restriction fragment length polymorphism. As a quality control six typical Candida strains from ATCC and 34 bacterial isolates were used. The yeasts cultures on Candiselect Ò 4 medium were incubated at 37 (C for 4 days. The colony morphology and pigmentation were read visually after every 24 h. Results: A pink-purple colonies were found in C. albicans and C. dubliniensis only. After 48 h a color of C. dubliniensis colonies became purple-bluish and differ from C. albicans, which were purple-violet with a pink halo around. The color and colony morphology of all strains of C. tropicalis, C. krusei and C. glabrata were in agreement with manufacturer description. Among the remain investigated Candida and non-Candida species the colorless or white colonies were very seldom and different shades of blue and green were observed. There was a problem with discrimination between C. glabrata and some C. inconspicua, C. parapsilosis, C. kefyr, and C. norvegensis. The sensitivity and specificity values for C. albicans were 98% and 87%, for C. tropicalis 100% and 98.4%, for C. krusei 100% and 100%, and for C. glabrata 100% and 90%. Conclusion: Candiselect Ò 4 could be regarded as a useful medium for presumptive identification of most commonly isolated Candida species, including fluconazole resistant.

In vitro activity of amphotericin B against Aleuriconidia derived from Aspergillus terreus C. Lass-Flö rl, A. Rief, A. Mayr (Innsbruck, A) Aspergillus terreus is an uncommon but emerging fungal pathogen, which causes lethal infections often refractory to amphotericin B (AMB). It has been suggested that aleuriconidia might be the source of AMB resistance. This study used aleuriconidia as inoculum and compared the minimal inhibitory concentration (MIC) of AMB and voriconazole (VRZ) to those obtained for conidia of 31 A. terreus strains using a broth microdilution method. In addition, we examined the in vitro susceptibility of hyphae derived from aleuriconidia and conidia of A. terreus. The MIC ranges of AMB for conidia and aleuriconidia were 1.25-5 lg/ml and MIC90 was 2.5 and 5 lg/ ml, respectively. The MIC ranges of VRZ for conidia and aleuriconidia were 0.25-1 and 1-2 lg/ml, and MIC90 was 1 and 2 lg/ml, respectively. The MIC ranges of AMB for hyphae of conidia and aleuriconidia were 1.25-5 lg/ml, and MIC90 was 2.5 lg/ml; the MIC ranges of VRZ were 1-2 lg/ml, and MIC90 was 2 lg/ml. In conclusion, our data confirm that AMB MICs from A. terreus aleuriconidia do not differ dramatically in comparison to MICs obtained from conidia. VRZ was significantly more active against aleuriconidia and showed consistently lower MICs.

Clinical evaluation of Aspergillus-PCR for detection of invasive aspergillosis in immunosuppressed patients C. Lass-Flö rl, G. Gastl, M.P. Dierich, A. Petzer (Innsbruck, A)

We evaluated the value of Aspergillus PCR as a tool for diagnosing invasive aspergillosis in patients at risk. Aspergillosis was assessed according to European Organization for Research and Treatment of Cancer/Mycosis Study Group definitions. Nine and 17 patients with proven and probable aspergillosis were evaluated. Whole blood samples prior (n=101) and during antifungal treatment (n=267), and tissue specimens (n=9) and/ or bronchoalveolar lavage fluids (n=17) were investigated. In patients with proven infections the sensitivities of PCR of lung samples were 100%, of blood samples prior treatment were 66%, during treatment 55%. Clearance of fungal DNA from blood was associated with resolution of clinical symptoms in 2 of 4 patients with proven infection. Consecutive positive PCR results for Aspergillus are associated with fatal outcome as 2 of 5 patients died. In patients with probable infections the sensitivities of PCR of lung fluids were 85%, of blood samples prior treatment were 57%, during treatment 42%. The benefits of PCR diagnosing and screening of whole blood is limited if sampling takes place once treatment has started. The performance of Aspergillus PCR from tissue samples should be recommended in addition to microscopic examination and culture technique for sensitive detection of fungal infection.

Identification of dermatophytes using partially digested ITS2-PCR Objectives: The routine identification of dermatophytes is a time-consuming process, depending on morphology. The forms of the spores are very informative, but this is mostly pending on the expertise of the researcher. The objective is to design a fast, easy and robust identification technique which allows the identification to species level from cultured dermatophytes. Methods: Internally transcribed spacer region 2 (ITS2)-PCR followed by fragment analysis (De Baere et al., 2001) has been shown to be a fast and reliable identification technique for yeasts. The used primers are universal for all fungi, so also molds and dermatophytes. The dermatophytes are however consisting of a variety of species of which some are highly related and ITS2length is identical. Therefore we added a partial restriction digestion to increase the discriminatory power.

Results: In total 29 species (each represented by 1-5 strains) were tested. For 16 of those species a unique fingerprint was obtained, which was different from all other species and which was constant for all members of that species, except for 3 species for which minor intraspecific differences were obtained. For other species we found different fingerprints, some of them unique, some also found for other species, which leads only for some members to a precise identification up to species level. Finally, a number of species had identical fingerprints so that final identification could not be obtained.

Conclusion: Partially digested ITS2 fragment lengths analysis enables the identification of dermatophytes up to species level (for most of the species) or to a cluster of 2-4 species (which are sometimes highly related by other methods as well). Observed discrepancies might in part be explained but not yet resolved taxonomy and may indicate the existence of not yet described species or the need for reclassification of strains. The technique can be seen as a fast identification technique for most of the routinely encountered dermatophytes. It worked especially well for the genera Arthroderma, Epidermophyton, Microsporon while Trichophyton species were more difficult to identify. Objectives: Victims of severe trauma are susceptible to opportunistic fungal infections such as zygomycosis, a rare but lifethreatening infection commonly caused by Rhizopus species. We describe the case of a child with necrotizing fasciitis caused by Rhizopus who was successfully treated with posaconazole (POS), an investigational triazole antifungal agent with in vitro activity against Zygomycetes. Case Report: A 12-year-old girl sustained bilateral haemorrhagic contusions of the temporal brain lobes, fractures of the skull and left tibia, and deglovement of the left leg after being hit by a truck. Drug-induced coma was initiated to control resistant intracranial hypertension. Initial treatment of the leg injury included almost daily removal of necrotic skin and muscle tissue and hyperbaric oxygen therapy. Thirteen days after the accident, Rhizopus spp and Trichosporon oviodes were cultured from multiple wound swabs. Subsequently, therapy with liposomal amphotericin B (LAMB) at 200 mg/d was initiated. Three days later, several areas of necrotic black ulcerations suggestive of invasive zygomycosis were observed, and the patient showed signs of sepsis. To avoid leg amputation, all suspected sources of infection were excised, daily hyperbaric oxygen treatment was given, and POS oral suspension (800 mg/d in divided doses) was added to the LAMB therapy. POS was given via nasogastric tube for the first 28 days and orally thereafter. After 2 days, most signs of sepsis disappeared, though the patient continued to experience fevers for the next 2 weeks. LAMB was discontinued after 9 days because drug-associated toxicity was anticipated. Necrotizing fasciitis responded gradually to antifungal treatment. Two weeks after initiation of POS, Rhizopus was no longer detected histologically or by culture of superficial tissue or muscle specimens. Objectives: Cryptococcus neoformans is an important pathogen in patients with impaired cell-mediated immunity and is the most common cause of fungal meningoencephalitis. C. neoformans is a facultative intracellular pathogen with several well-defined virulence factors that include the production of a polysaccharide capsule, melanin and growth at 37°C. Identification and characterization of enzymes produced by C. neoformans are important because of their potential role in virulence, induction of host immune response and for epidemiological typing.

Methods: Thirty-nine strains were included in the study, 29 obtained from patients with AIDS and 10 from patients with lymphoprolipherative diseases. The enzymatic profiles of C. neoformans isolates were obtained by using the API-ZYM system (bioMérieux, France) as a whole-cell assay with an inoculum of 106 cells/ml. The phospholipase activity was tested using the egg-yolk plate method. The ratio of colony diameter to the diameter of the colony plus the pericolonial precipitation zone on agar (Pz) correlated with phospolipase activity. Results: All tested strains were identified as grubii or neoformans variety by standard methods. The API-ZYM system is a screening test for the activity of 19 enzymes. Eleven of the 19 enzymes were found to be positive and no significant difference between two groups of isolates was observed. The activity of butyrate esterase was detected in 38 strains and activities of caprylate esterase lipase, leucine arilamidase and acid phosphatase were registered in 37 isolates. The activities of following enzymes were negative: lipase, cystine arilamidase, trypsin, chymotrypsin, alpha-galactosidase, N-acetyl-beta-glucosaminidase, alpha-mannosidase and alpha-fucosidase. Seventeen enzymatic patterns were identified according to the profiles obtained by API-ZYM system. Only one strain, isolated from an AIDS patient, was detected as phospholipase negative. There was no significant difference in phospholipase production between isolates obtained from patients with AIDS and lymphoprolipherative diseases (mean Pz, 0.52 vs. 0.55; p>0.05). Conclusion: We found no difference in the enzymatic activity between strains infecting two different groups of immunocompromised patients. Each enzymatic activity has its own pathway, which could be separately related to C. neoformans virulence, and it was recently demonstrated that extracellular phospholipase activity is a virulence factor for this fungus.

The epidemiology and antifungal sensitivity results of nosocomial infections caused by Candida species Of all patients 77.5% and 76.2% of patients with candidemia were hospitalized in intensive care units. C. albicans (50.6%) the most prominent Candida species was followed by C. glabrata (16.3%), C. tropicalis (16.3%), C. parapsilosis (10.0%), C. krusei (3.1%), C. lusitaniae (1.2%) and C. kefyr, C. sake, and C. pulcherrima. All isolates were sensitive to amphotericin B (MIC ‡ 1). Fluconazole resistance was 4.3% and 15.6% for all C. albicans and other species respectively. Resistance to itraconazole was found to be higher, which was 11.4% for C. albicans, and 26.5% for other species. Cross-resistance to itraconazole was determined in 12 of 13 (92.3%) isolates which were resistant to fluconazole.

Conclusions: The high resistance to azoles which can be considered as indicator of inappropriate antifungal usage should be paid attention. Empiric antifungal treatment by azoles which is routinely used by clinicians without differentiation between infection, colonisation and contamination, especially for Candida species isolated from urinary tract, threatens the efficacy and safety of antifungal therapy. Antifungal sensitivity tests should be used to determine the regional antifungal sensitivity patterns, to follow up of the patients with invasive infections like candidemia who need long term therapy and for determining the resistance and to choose alternative antifungal drug for those unresponsive and have recurrent mucosal disease.

Tropical and parasitic diseases Objectives: Loiasis is a nematode parasitic infection endemic to sub-Saharan Western Africa. Native people from endemic areas emigrated to developed countries are often infected with this worm. We report a case of conjunctival loiasis in a young black male born in Guinea. Case report: Patient was living in Spain for the last 2 years. He complained of eye pain and noticed a Ôworm passing through the left eyeÕ. He was attended in the ophtalmologic emergency care. Moderate eosinophilia (21%) was present in peripheral blood. Inflammatory reactions (Calabar swellings) were not present. After surgical and medical treatment, the patient was discharged from the hospital. At the present time, 10 months latter, he remains asymptomatic.

Results: The presence of migrating subconjunctival worm was observed, and removed surgically. It was identified as a female adult of Loa loa (55 · 5 mm). In blood samples collected during day-time, 300 lm long microfilariae were observed in direct wet smear. Hematoxylin stain showed the external sheath and Giemsa stain the nuclei extended to the end of the tail. Microfilariae discharged by the female worm were preserved in destilled water in the laboratory at room temperature. On day seven both free forms and eggs were present. Haemonchosis, a disease caused by Haemonchus contortus is of considerable economic importance in sheep cattle and goats throughout the world. Severe infections in sheep may cause considerable morbidity and mortality rates; even more, moderate infections in lambs, may give rise to body weight reduction and in adults cause various degrees of anaemia and decrease the animal products. Control depends on repeated application of anthelmintics and therefore there have been many attempts to develop a vaccine against this disease.In a trial, in Ahwaz area, the ability of an experimental vaccine made from the whole gut homogenate of Haemonchus contortus was evaluated. A group of 5 Arabian female lambs were immunized twice with 100 lm whole gut homogenate (WGH) of Haemonchus contortus diluted in 1 ml PBS and emulsified in 1 ml Freund's adjuvant. The other group (5 Arabian female lambs) received 1 ml PBS emulsified in 1 ml of the same adjuvant. The first immunization was performed intra muscularly in the semi-membranous muscles of each hind leg. Booster immunization administered subcutaneously on day 21. On day 33 each lamb was challenged with approximately 10,000 Haemonchus contortus third-stage larvae. From the first immunization until challenge the animal were bled with an interval of 10 day and then weekly from challenge until the end of study. Sera were tested by ELISA and western-blotting. Lambs were necropsied 6 week post-challenge for recovery of nemetodes.Vaccinated lambs showed a 77% reduction in mean EPG, 75% reduction in mean male worm numbers, 80% reduction in mean female worm numbers and 78% reduction in mean total worm numbers. A significant difference in means optical density of sera in ELISA was noticed (p = 0.001). Proteins of Hemonchus contortus gut were seperated by SDS-PAGE. Then sera of vaccinated group indicated a high reactivity in western-blotting.

Clinical features and therapy of trichinosis in Belarus 

The likelihood of the clinical diagnosis was enhanced by both anamnestic and epidemiological data, and serological confirmation was obtained by testing IgM with ELISA. 90% of the patients presented with a typical clinical picture of trichinosis, including fever, periorbital edema, myalgia (usually affecting the gastrocnemius muscle), and eosinophilia in the first days of disease. However, 5% of the patients showed eosinopenia (down to 2%) within the first days, which correlated with the severity of the disease. The clinical features of these latter patients also included myocarditis, neurologic disorders and respiratory failure.Treatment of trichinosis is known to be not satisfactory. In prescribing the antiparasitic therapy, attention should be paid to the phase of the disease, its severity and the eosinophyl blood count. Our approach was highly individualised and consisted of both etiologic and pathogenetic (or symptomatic) therapy. The etiologic therapy aimed at destructing intestinal worms, preventing the development of cyst walls around the larvae, and destructing the Trichinella larvae in the muscle fibers. We used mebendazol 0.3 g every 8 h for 10 days. This etiologic therapy was most effective in the incubation phase or in the first days of disease, when localisation of Trichinella still remained intestinal. Vice-versa, therapy with mebendazol during the muscular period of disease not only was less effective but could also lead to a further exacerbation of the symptoms. The symptomatic therapy included antihistaminic and non-steroid anti-inflammatory drugs. In the most severe invasive forms, we used glucocorticosteroids (prednisolon 20-80 mg/day for 5-7 days).

Intestinal parasites in native and foreign citizens of Athens during a two-year period V. Skandami, C. Kotsi, C. Achilleos, C. Kontou-Castellanou (Athens, GR)

Objectives: To determine the incidence of intestinal parasites in citizens of Athens and analyze the difference between the natives and immigrants coming from developing countries. Methods: During a 2-year period (2003) (2004) , 1680 feacal samples (1399 from natives and 281 from foreigners) were examined in our hospital laboratory for the presence of parasitic pathogens. Of them, 592 were treated in the hospital and 1088 were outpatients. The 86.9% of outpatients were workers who were examined in order to obtain a health clearance certificate. All specimens were examined in direct microscopy using wetmount and formalin-ether concentration methods. Results: Of the 1680 specimens examined, 24 (1.4%) were found positive for pathogen intestinal parasites: Giardia lamblia 15, Enterobious vermicularis 5, Heterophyes heterophyes 1, Ancylostoma duodenale 1, Entamoeba histolytica/dispar 2. In 15 specimens (0.9%) Blastocystis hominis were detected and in 2 non pathogenic Amoebae (Entamoeba coli and Iodamoeba butschlii). The incidence of pathogen parasites was 0.7% for the Greeks and 5.0% for the foreign population.

Conclusion: The frequency of intestinal parasitic infection in Greece, is relatively low but not rare, especially during the recent years because many immigrants coming from developing countries have introduced new parasites. The alertness of public health services is critical in order to prevent parasite spreading from the carriers to the healthy population.

Control of coccidiosis in poultry K.K. Hayat (Dera Ghazi Khan, PAK)

An egg adapted gametocytes, E. tenella, vaccine(s) were used against coccidiosis in chickens. On day five chicks were divided into four groups and administered vaccine(s) orally viz; Vaccine I (gametocytes), Vaccine II (gametocytes inactivated), Vaccine III (gametocytes sonicated inactivated) and fourth group was served as control given normal saline. On day 15 immunized chicks were challenged with 60,000-70,000 sporulated oocysts of mixed species of Eimeria. A total of 126 birds were used in this study out of them 94 birds were scarified and 32 chicks died during challenge response. On day 21st post vaccination birds were subjected to postmortem and their lesions score were recorded. A maximum of 46 birds having lesions in intestine and caeca of Group-IV birds were observed while a minimum of 17 birds having lesions in intestine and caeca were observed in Group-III. There was non-significant difference (P > 0.05) in lesions score of Group-I, II and IV. Lesions scores in Group-III were significantly different (P > 0.05) from Group-I, II and IV. Cellular immunity was studied in the course of immunization of animals and during their conseguent infection with Entamoeba histolytica, 81 reedleswe albino rats being employed. 4 strains of polyxenic culture Ent.histolytica were used isolated from patients and carriers. For immunization of rats, the killed antigen composed 50,000 trophozoies per 1 ml of the medium was administered subcutaneously 5 times at a weekÔs interval.

A week following the immunization the rats were infected through caecum with 0.5 ml of the live culture containing about 100,000 trophozoites. On days 6-9 p.i. all the rats appeared to be infected. On days 14-21 and 28 p.i.(the end of the observation ), 93 and 97% of infected animals were registered respectively. The control animals were not infected. Up to the 6th day, 75% of young rats were infected. In most of the infected adult (91.3%) vegetative forms of Ent.histolytica were found in the caecum and moderate morphological changes were observed in 72.8% of these (hyperemia, oedema, haemorrhage, ulcer) . In the submucous membrane of almost all the experimental animals vascular perivascular mononuclear infiltration was observed. With the help of the migration inhibition reaction of macrophages, it has been established that in the process of immunization and subseguent infection lymphocytes sensible to amoebic antigen appear in the rat's body. The danger the observation the higher the degree of body sensibilization. The evidence presented shows that the cellular mechanisms of immunity the of great significance in the pathogenesis of amoebiasis. Results: The inflammatory diseases of genitals were preceded and accompanied in half of the patients of leiomyoma. In 60% various combinations of pelvic inflammatory disease. The method ''the case-control'' has revealed a high risk of development of a myoma of an uterus (the relative risk of occurrence by a parameter of the attitude of chances is 1.75) in patients with inflammatory diseases of genitals. The bacteriological research of 80 myomatous units, has revealed the associated microbic flora in them, in comparison with non changed fabrics of a uterus. Identification of flora by carrying out 400 PCR testifies to presence Chlamydia trachomatis (mainly in submucous) and Ureaplasma urealyticum (in intermuscularsubserous) myomatous units, even at their absence before operation in underlying departments of sexual ways. Pathohistological research of non struck with a myoma uteruses, removed concerning endometritis in a combination with purulent tubo-ovarian formations in three women of 24-25 years old, has shown formation of the reclaiming rudiments consisting from proliferated, concentrically located smoothly-muscular cells (SMC) around the inflammatory infiltrates in myometrium-leiomyomatoz. Immunohistochemical researches with PCNA have established its accumulation in nucleus SMC in inflammatory mononuclear infiltrates zones in myometrium at endometritis, that is considered by us as the trigger of a myoma of an uterus. The conclusion: In our opinion, uterin fibroid embolization (UFE) is a prime method of a choice in treatment of a myoma of a uterus. In some cases there is a risk of secondary infection in degradation of unit or allocated myomatous detritus, that can demand hysterectomy. Taking into account all set forth above, before carrying out UFE, in our practice, we used: Fromilid 500 mg twice a day during 10 days + Tiberal 500 mg twice a day during 10 days. Using the given circuit, in complex treatment of a myoma of an uterus, has allowed to increase efficiency of conservative treatment in twice.

Comparative evaluation of effectiveness of urogenital clamidiosis treatment with antibiotics and ozonotherapy N. Sarapulova, N. Guryanova, T. Kolesova (Perm, RUS) Objectives: The aim of this study is to examine the comparative evaluation of urogenital clamidiosis treatment with antibiotics and ozonotherapy in women of childbearing age. Methods: were two groups of women of childbearing age from 20 to 45 years old with urogenital clamidiosis under investigation. I group of 37 patients were treated with classical antibiotics treatment regimen. II group of 31 got ozonotherapy as intravenous introduction of ozonized physiological solution 200 ml with concentration 2-3 mg/l, N°. 7-12 and ozonized oil was used locally. Estimation of the therapy results in both groups was held twice with the month interval with the use of enzyme immunodetection (EI) (diagnostic antibody titre 1:32 and more) and direct immunofluorescence (DI Objectives: Direct detection of microbial DNA in clinical samples like blood or cerebrospinal fluid (CSF) requires a reliable DNA isolation procedure and a sensitive PCR amplification, due to low microbial loads in these clinical samples. In this respect optimal recovery of target DNA during extraction is necessary. In this study we compared the performance of DNA recovery of the automated Roche MagNA Pure Total Nucleic Acid Isolation Kit (M-extraction) and the manual Boom extraction method (Si-extraction).

Methods: DNA recovery experiments were conducted with PBS, CSF, plasma and whole blood (4-8 replicates for each sample matrix) for both methods. Other reconstruction experiments were performed with positive control DNA's for EBV and VZV (2 dilutions, tested in 5-fold) in PCR negative blood and analysed by PCR. Also serial diluted CMV was spiked in CMV negative blood (4 dilutions tested in 8-fold) and analysed by CMV PCR. Finally clinical blood samples were tested for EBV (n = 23) and CMV (n = 16) by both methods.

Results: Recovery experiments showed reduced recovery for M-extraction (~20%) compared to Si-extraction (>80%) for all matrices tested. Part of the missing DNA from the M-extraction could be retrieved from binding (~20%), washing (~5%) and silica matrix (~15%), leaving~40% of the DNA irretrievable. Positive control DNA's showed between 7.6 and 14.2 and 1.9 and 7.9-fold lower PCR signals after M-extraction, compared to Si-extraction for EBV and VZV respectively. PCR analysis of spiked CMV showed reduced recovery and loss of sensitivity for M-extraction, resulting in 0% (0/8) and 88% (7/8) hit rates for the lowest spiked CMV loads, whereas Si-extraction obtained 50% (4/8) and 100% (8/8) Objective: New therapeutic options have led to considerable improvements in viral infection management and it is also clear that such treatment requires careful consideration and followup. Monitoring of viral load in immuno-compromised patients applying a pre-emptive disease management strategy is important to manage opportunistic infections and prevent disease. In order to meet the growing clinical demand for these patients, three sensitive assays have been developed for measuring CMV, EBV and HBV viral DNA in serum, plasma or whole blood. Method: Real-time PCR quantitation utilising the Scorpionä technology provides sensitive and rapid methods for determination of CMV (affigene Ò CMV trender), EBV (affigene Ò EBV trender) or HBV (affigene Ò HBV trender). An internal control (IC) is included in each sample to indicate for the effects of inhibition, and control for sample preparation, amplification and detection. A manual sample preparation protocol with input volumes from 50 to 1000 ul (serum, plasma or whole blood) is used.

Results: Performance testing demonstrated: affigene Ò CMV trender: assay sensitivity (95% positivity-rate) of~100 copies/ mL and at least 6-log dynamic range for CMV clinical specimens. Equivalent quantitation of CMV strains has been shown using clinical specimens.affigene Ò EBV trender: assay sensitivity (95% positivity-rate) of <500 copies/mL and at least 6-log dynamic range for EBV clinical specimens. Equivalent quantitation of EBV genotypes A and B has been shown using clinical specimens. affigene Ò HBV trender: assay sensitivity (95% positivity-rate) of <4 U/mL and a 8-log dynamic range for HBV clinical specimens. Equivalent quantitation of HBV genotypes A-H has been shown using clinical specimens and quantitated genotype plasmids. Normal blood donors were negative for HBV by serology. No cross-reactivity for potential cross-reactive pathogens has been observed in any of the assays. A 2-month-old female infant was admitted to the paediatric clinic because of progressive and unexplained jaundice and light coloured stools. Symptoms began 20 days before admission. On examination, pediatricians found distened abdomen, hepatomegaly and enlargement of the spleen. The ultrasound test detected a tiny gall bladder, while during the HIDA scan test the radioactive dye could not flew through the billiary system. Blood tests showed the following: PT and PTT times were prolonged, repeatedly blood cultures were negative and serologic tests ruled out an acute infection with A, B, C and E hepatitis viruses, HIV, HSV, VZV, EBV, Rubella viruses, Toxoplasma gondi and Coxiella burnetii. CMV serologic study was based on the perfomance of ELISA ( DiaSorin s.r.l.) and IFA ( PANBIO, inc ) methods. Sera samples were collected on the 1st, 5th, 8th day after admission. CMV IgG antibodies were found positive by ELISA test (54 Au/ml, 60 Au/ml, 66 Au/ml, respectively ). In all the tested samples CMV IgM antibodies were found positive by both ELISA (+) and IFA tests ( 1:40, 1:80, 1:80 respectively ). At the same times, PCR analysis was carried out (COBAS AMPLICOR CMV MONITOR test) and detected CMV-DNA in blood and urine samples. Only the first blood sample was positive (5.2 · 10 2 copies/ml), while two urine samples were CMV positive (3.45 · 10 3 , 2.69 · 10 4 copies/ml). The baby's mother was CMV-IgG (+) 236 Au/ml and CMV-IgM (-). CMV-DNA has not been detected in her blood and urine samples. Under antiviral treatment with ganciclovir first the blood on the 5th day, and then urine samples on the 8th day became negative. However, this did not affect the progressive damage of the liver. Portal hypertension and hepatic coma eventually occurred. Conclusion: CMV perinatal or congenital infection may participated in the etiopathogenesis of the the neonatal cholestasis. The cause is difficult to be determined, since there was no history of maternal CMV infection and the serology of the mother was unknown before and during pregnancy and at the time of the birth. At that time, the serology of the infant was also unknown and CMV-DNA was detected 2 months later. Enterovirus RNA was detected through RT-polymerase chain reaction. Sixty three clinical records were revised. Results: Isolation of Enterovirus in CSF was positive in 65 of 926 of the samples studied in our laboratory (7.0%). Forty-eight (74%) of these patients were males and 17 were females (26%). Most patients were under 14 years and 11 of them (17.2%) were infants younger than one year. Enterovirus was isolated only in three adults (4.8%).The evolution time until hospital admission was less than 24 h in 57 cases (90.5%) and longer than two days in 6 patients (9.5%). Hospitalization was required in all the patients. Six of them were discharged before 48 h (9.5%) and 26 remained in the hospital more than 5 days (41.2%).The most common symptoms were fever, headache and meningeal symptoms. Antibiotic treatment was given to 39 patients (61.9%) and only in 6 cases (9.5%) it was removed before of the fourth day. The rest of the patients received antibiotic treatment until viral culture result was known. The evolution of the patients was favourable without sequels.

Conclusions: Enterovirus are the most important cause of viral meningitis in our environment with a high incidence in infants.

A seasonal distribution has been found in Enteroviruses meningitis; the highest incidence was recorded from May to June (Spring). The knowledge of this seasonal distribution is a diagnostic indicator, which can help to predict infection in these months and adequate the treatment.

Telomerase activity in cutaneous and anogenital warts: a preliminary study Objective: To evaluate telomerase activitiy in cutaneus and anogenital warts. Methods: Normal skin, genital and cutaneous lesion specimens were collected from 6, 9 and 14 patients, respectively. Telomerase activity was detected by classical immunohistochemstry by using telomerase catalytic unit (hTERT) antibody. Specimens were stained with monoclonal antibody (Novocastra). Results: Staining pattern was located in basal and suprabasal location in normal skin specimens. However, telomerase activity in basal, supra basal and spinose layers was detected in all anogenital wart specimens but this staining pattern was detected only 7of 14 cutaneous specimens. Mean telomerase activity of the specimens was 66.9%, 61.7%, 27.6% in anogenital wart, cutaneus wart and normal skin specimens, respectively. No association in telomerase activity was determined between anogenital and cutaneus warts. The telomerase activity was significantly higher in HPV infection groups (cutaneus and anogenital) than in normal skin. Conclusion: The increase in the activation of telomerase by HPV found in this study is consistent with all other proliferative lesions. 3 (4-810) . Only 12 (19%) were receiving highly active antiretroviral therapy (HAART) and 49 (78%) had also HVC infection. The onset was acute in 43 cases (68%). All the patients were febrile, 48 (76%) had respiratory symptoms and 33 (52%) cardiac murmur. Chest X-ray was normal only in nine cases (14%) and septic embolisms were observed in 28 (44%). The vegetations were detected by two-dimensional echocardiography and tricuspid was the most frequent affected valve (53 cases; 84%). Mitral valve endocarditis was diagnosed in five cases (8%), aortic in 6 (9.5%) and pulmonary in 3 (5%). There was valvular insufficiency in 39 cases. Blood culture was negative in 28 episodes (44%), there was three cases of polymicrobial IE (4.8%) and S. aureus was the most frequently isolated organism (19 episodes; 30%). Patients were treated with antibiotics during 2-4 weeks, but 7 (11%) needed treatment for 6 weeks. Therapy was depended on the organism's susceptibility but initially the subjects were empirically treated with cloxacillin with or without tobramycin. 

Temporal pattern of infectious complications in HIV-infected patients era and their consequences on continued anti-HIV and antimicrobial therapy. The antiretroviral management and the indication to gabexate and/or octreotide administration in the different clinical-laboratory situations, deserves extensive, controlled studies.

Occurrence of cholangiocarcinoma after AIDS-related cholangitis survival was 3.4, and maximum years of survival was 11. Thirtyone of the patients died, of those 26 died at their first admission. The most common professions were, drivers and workers. Eighty per cent of the women were housewives. Only one of the patients was university graduated. Heterosexual intercourse was the most common (86%) route of transmission. Blood transfusion, and IV drug use was the other routes. One male patient declared men with men sexual relation at the first interview, and 5 did after more detailed interview. Median CD4 count at the diagnosis was 198 (min 5, max 1051), and the mean viral load at the diagnosis was 122, 000 (min 400, max 107), 36% of the patients were grouped in C3 category at their first admission. The most commonly detected opportunistic infections were Candidal infections ( Results: Sustained virologic response (defined as an undetectable serum HCV RNA level 24 weeks after treatment was completed) was not achieved in any of 12 patients treated with interferon-alfa alone. Combination therapy with interferon-alfa and ribavirin was associated with better results: 4/10 (40%) patients still untreated with interferon-alfa, 2/4 (50%) relapsers, and 2/7 (29%) non-responders to previous interferon-alfa monotherapy achieved sustained virologic responses. Combination therapy with pegylated interferon and ribavirin has been used in 3 patients. Sustained response was achieved in one patient who had relapsed after treatment with interferon-alfa and ribavirin and in 1 of 2 non-responders to this combination therapy. There were no serious adverse events and it was not necessary to reduce dosages or even cease therapy prematurely. Conclusions: The efficacy and tolerability of antiviral treatment in haemophiliacs did not differ from other patients with chronic hepatitis C.

Prevalence of hepatitis B virus genotypes in chronic infected patients A. Suárez, E. Vazquez, C. Sánchez, J.J. Picazo (Madrid, E)

Objectives: Hepatitis B Virus (HBV) genotypes have been associated with specific patterns of liver disease and treatment response. The aim of this study were to determine the prevalence of HBV genotypes, serum HBV-DNA level and severity of liver disease in chronic infected patients.

Methods: A total of 87 HBV infected patients were enrolled, 63 males and 24 females. The mean age was 46 years (range 28-77 years). Fourteen of them were co-infected with human immunodeficiency virus (HIV). Sera were tested for HBV genotyping by reverse hybridization line probe assay INNO-LiPA HBV Genotyping (Innogenetics) after a PCR sensitive amplification protocol. Quantitative HBV-DNA levels were determined using Cobas Amplicor Monitor kits (Roche). Detection of Hepatitis B e antigen (HbeAg) in patient serum samples also was performed using the ETI-EBK enzyme-linked immunosorbent assay (DiaSorin). Results: All HBV genotypes (A-G) were found. The genotypes A, D, E and F were common and constituted 22%, 36%, 8% and 12% of the total patient population. Genotypes B, C and G were uncommon and represented less than 2% each one. Fifteen patients were infected with two HBV genotypes. On the HIV coinfected patients genotypes A, D and mixed (A + G) were prevalent. Only in 14 of the 87 patients was detected the presence of serum HbeAg, ten of them were HIV-infected, and it was associated with HBV genotypes A (30%), D (20%) and mixed genotype A + G (30%). Among the patients HbeAg negative genotypes D, A and F were more frequent 37%, 20% and 12% respectively. Analysis of baseline serum HBV-DNA levels in HBeAg positive patients revealed that were higher than 106 excepted in two of the three mixed genotypes. Genotypes B, C and G were associated with high levels of serum HBV-DNA. The lower level of serum HBV-DNA was detected in a patient with genotype D and negative HbeAg. Conclusion: Our data indicate that HBV genotypes A, D and mixed A + G were associated with a higher prevalence of HbeAg, and HIV-infected patients. Genotypes B and C presented higher serum HBV-DNA levels in HbeAg negative patients. Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 Methods: RNA extraction was done on serum sample from an IVDU who was chronic HBsAg carrier and anti-HD ELISA positive. Twelve pairs of primers were used to amplify six partially overlapping fragments, covering the entire HDV genome by RT-nested PCR. PCR products were purified and subjected to automatic cycle-sequencing procedure. Editing of raw data was accomplished by the Generunner and Chromas softwares. Alignment analysis of complete genome and HDAg amino acid sequences were carried out on IR-1 and eleven reference sequences. These analyses were done by ClustalW method, ClustalX software and subjected for phylogenetic analysis by applying neighbour-joining model for tree construction. Results: The majority of patients (90%) showed infection with genotype D, genotype A was the second most common (9%) and only 1 patient had mixed type (D and G). Polymerase mutants were identified in 49 patients (78%).The M204V/I mutant was observed in all (100%) YMDD variants of both genotypes D and A. The L180M mutant was detected in 4/43 (8%) patients with genotype D and in 3/5 (60%) patients with genotype A. There were no patients in whom mutation V/L/M207/I occurred. HBV-DNA was detectable in 64% of patients (baseline > 35 copies/ml). The prevalences of variants and viral loads were respectively the following: low viral load (10 2 -10 3 copies/ml) 25% mutants 75%, medium viral load (10 4 -10 5 copies/ml) 30% mutants 60%, high viral load (>10 5 copies/ml) 45% mutants 90%.

Conclusions: Genotype D is the most common in the area of Northern Greece. The M204V/I mutation is the most frequent among genotypes D and A. Although the appearance of mutants seems to have no correlation with HBV genotypes and viral load, the type of change is propably relayed. The test was conducted to find infection of Hepatitis A virus (HAV) and the genotypes against twenty-six of the patients with fatigue and nausea. Fifteen isolates (57.7%) were amplified by RT-PCR using HAV specific primer, and then compared to 168 bp nucleic acid sequences in VP1/2A junction region for the genotypes. Based on 79% similarity, 13 out of 14 isolates were clustered with genotype IA and 1 out of 14 isolate was separated to IIIA. Above 13 isolates found in genotype IA were sporadic to Japan, China, and Korean strains which are grouped determined by it's geographic region with over 95% similarity. The history of patients informed the high relationship of endemic or family transmission, and it also showed the changes according to risk age group followed by the economic growth as 14~46 years-old patients.

Secondary acute viral hepatitis (2003). The etiological diagnosis was established on clinical and paraclinical data (serological markers -ELISA method). In order to establish a complete diagnosis we checked the following syndromes: cytolitic, hepatoprive and billiary retention. Also we studied parameters such as age, sex, residence and period of hospitalization.

Results: From the total of 53 patients the most of the cases with secondary acute viral hepatitis were patients with infectious mononucleosis -25 cases, followed by patients with rubella -17 cases, by patients with cytomegalovirus infections -9 cases, and by varicella zoster -2 cases. Patient's age was between 2 and 17 years. Sex ratio shown a predominant feminine injury (M:F = 1:2.53). For residence point of view the majority of children were from urban area (31 patients from 53). The cytolysis syndrome was presented with a slight elevation of ALT (2-5 N) in majority of cases (except one case with infection with a severe form of hepatitis), such as billiary retention syndrome. The hepatoprive syndrome was light and medium in all cases except the patient already mentioned with varicella zoster infection in an immunodepressed person. There were no colestatic forms and no chronically cases or deceases. Conclusions: The secondary acute viral hepatitis presented a favorable evolution in almost all our cases. In the context of some viral diseases hepatic injury is frequent but slight, sometimes asymptomatic. In these diseases generally the liver affection is underestimated in our area. 

Response to interferon-alpha in chronic hepatitis B with and without precore mutant strain S. Akhan, E. Azak Karali, Z. Yulugkural (Kocaeli, TR)

Objectives: Chronic hepatitis B is an important public health problem worldwide and in Turkey. In Turkey chronic hepatitis B were mostly precore mutant strains. We investigated whether the presence of precore mutant affects the response to interferon-alpha in patients with chronic hepatitis B. Study: Chronic hepatitis B patients who were admitted to the Infectious Diseases and Clinical Microbiology polyclinic in the last eight months were included in this study. Thirteen with hepatitis B e antigen-positive, hepatitis B e antibody-negative and ten with hepatitis B e antigen-negative, hepatitis B e antibody-positive (precore mutants) chronic hepatitis patients were treated with natural interferon-alpha 24 months. Before treatment, all patients had liver biopsy to grade and stage liver disease. Liver biopsy showed chronic hepatitis (necroinflammatory score 4), there was no cirrhosis and nobody had interferon therapy before. Serum HBV DNA level criteria to begin the therapy was 104 copies/ml for precore mutant strains and 105 copies/ml for non-mutant strains. Results: Serum HBV DNA level before treatment was not different between the two groups (p = 0.053). At the end of treatment, serum HBV-DNA was decreased to undetectable levels in 30% of mutant group and 23% of the non-mutant group. The decrease of serum HBV-DNA level was found significant (z = )2.66; p = 0.008) in mutant group and not significant (z = )1083; p = 0.279) in non-mutant group. Six months after treatment, the percentage of cases with a decrease in the transaminase level to Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 within the normal range was significantly higher in mutant group (p = 0.028) than in non-mutant group (p = 0.108).

Conclusions: Chronic hepatitis with precore mutant strain seems more responsive to IFN-alpha. The reason of not expecting result may be the serum HBV-DNA of four patients in non-mutant group showed increase in spite of the treatment and none of the patients in the mutant group.

Neutrophil oxidative metabolism in children with chronic hepatitis C T. Wozniakowska-Gesicka, M. Wisniewska-Ligier, P. Lewkowicz (Lodz, PL)

A growing number of data suggest the role of reactive oxygen species (ROS) in the pathogenesis of chronic type c hepatitis. Activated neutrophils, macrophages and Kupffer cells are basic sources of ROS in the course of the inflammatory process in the liver. The aim of the study was to assess the metabolism of neutrophil oxygen in children with chronic hepatitis C.

Material and methods: The study comprised 14 children with chronic hepatitis C (group I), 4 children with positive serum anti-HCV antibodies and negative HCV-RNA as well as normal ALT activity (group II) and 6 healthy children 9 (group III). Production of reactive oxygen species by neutrophils using the method of chemiluminescence (CL) with luminol (MLX Microtitier Plate Luminometr, Dynex, USA) was assessed. We estimated chemiluminescence response (CL max and CL total) of neutrophiles both unstimulated and stimulated by formyl-methionyl-leucylphenyloalanine (fMLP), opsonized zymosan (OZ), phorbolmyristate-acetate (PMA) without and after priming with tumor necrosis factor alpha (TNF-?) was estimated.

Results: CL max f MLP stimulated without preactivation was significantly lower in group I and II than in group III (p < 0.04, p < 0.06, respectively). CL max OZ stimulated was significantly higher in group I in comparison to group II (p < 0.04) and it tended to be higher in group I than in group III. CL max PMA stimulated was lower in group I compared to group III (p < 0.08). CL total fMLP stimulated was lower in group II (p < 0.07) compared to group I and it tended to be lower in group I and II compared to group III. CL total OZ stimulated was significantly higher in group I than in group II (p < 0.05) and group III (p < 0.03). CL max and CL total OZ stimulated and preactivated with TNFa was significantly lower in group I compared to group III (p < 0.02 and p < 0.04, respectively). Summing up: In the course of chronic hepatitis C in children, both neutrophil preactivation and inhibition of neutrophil release of reactive oxygen species, which occur as a result of different mechanisms, are observed.

One year combined treatment with ribavirine and intron A in children with chronic hepatitis C T. Wozniakowska-Gesicka, J. Kups, M. Wisniewska-Ligier (Lodz, PL)

The aim of the study was to assess the effect of 12-month therapy with IFN-a and Ribavirine in children with chronic hepatitis C.

Material and methods: The study comprised 22 children treated with Intron A administered subcutaneously at a dose of 3 M.U. Three times a week and Ribavirine orally 15 mg/kg body weight daily forthe period of 12 months. In the course of the treatment, ALT activity, haemoglobin, leucocyte and thrombocyte were determined. HCV-RNA in serum was performed at the beginning, after 6 month and after the therapy. Results: After 12 months of combined treatment with IFNalpha and Ribavirine the elimination of HCV-RNA and normalisation of ALT activity were observed in 14 children. In 2 children the normalisation of ALT activity without elimination of HCV-RNA was observed. Six children did not respond to 12month combined therapy. A lower mean activity of ALT at the beginning of the treatment was observed in children who fully responded to the combined therapy as compared to those who did not respond to the treatment. The following side effects were found in all the treated children: fever, headache, abdominal pain loss appetite, hair loss, myalgia and hypersomnia. In 12 children transient leucopenia, in 6 thrombocytopenia and in 5 anaemia were detected. Conclusions: 1. Full response to the combined therapy (HCV RNA elimination and ALT normalisation) was obtained in about 64% of the treated children.

2. Numerous side effects observed in the course of the combined therapy did not cause the interruption of the treatment. Objective: Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi sarcoma. HHV-8 has been associated to other diseases such as primary effusion lymphoma, Castleman disease, bone marrow failure and oral cavity plasmablastic lymphomas (PBL) in HIV+. Epstein-Barr virus (EBV) is known as a cofactor in AIDS-related lymphomas. We report the case of an HIV+ male which presented with two HHV-8 related neoplasms: a colonic PBL and a sigmoid Kaposi sarcoma. To our knowledge this is the first case of colonic PBL in the literature, as well as the first case where these two malignancies are associated. Case: A 41-year-old male had a 3-month history of liquid diarrhoea, fever and malaise. He was known HIV+ for 11 years and had not previously received HAART. Physical examination was normal except for a facial lipoatrophy and small cervical lymphoadenopathies (<1 cm). Hemoglobin 9.9 g/dl, WBC 5030/mm 3 , platelets 227000/mm 3 , ESR 29 mm/h, creatinine 0.89 mg/dl, GOT 61 U/l, GPT 55 U/l, LDH 545 U/l, beta-2microglobuline 3.58 mcg/ml, CRP 4.82 mg/dl; CD4 135/mcl, HIV-1 viral load 302,000 copies/ml. PCR for EBV in the cerebrospinal fluid was positive. A colonoscopy showed a purplish lesion in the sigmoid colon and an ulcerated mass in the caecum; both were biopsied. The first one was informed as a Kaposi sarcoma and the second one as a PBL. A bone marrow biopsy showed lymphomatous infiltration. D4T, 3TC and lopinavir/ritonavir was started, as well as CHOP chemotherapy. The latter was stopped after the third course due to progressive deterioration. The patient developed a palsy of the III and VII cranial nerves. Opportunistic infections were ruled out and a spinal tap revealed lymphomatous meningeal involvement. He died 6 months after the initial referral.

Conclusion: HHV-8 is involved in the pathogenesis of both malignancies, Kaposi sarcoma and PBL. Nevertheless, neither this association nor the peculiar localization of the PBL (right colon) have been described. Only few other extra-oral sites have been found: nose, lung, mediastinum, stomach, testicle, bone and ano-rectum. Diagnostic procedures with regard to HHV-8 infection are not routinely performed. Repeated detection of HHV-8 has been described to increase the risk of active Kaposi sarcoma. We hypothesize that extra-oral PBL might also be related to HHV-8 as a cofactor, similar to EBV. We suggest that a better understanding of the mechanisms of the infection could provide prevention and treatment strategies. 

The prevention and treatment of neonatal herpesviruses infections D. Bartosova (Brno, CZ)

Objectives: In this report, the authors have presented clinical picture, course and therapy used in 13 infants with congenital VZV infection, 12 infants with congenital herpes simplex infection and 18 infants with CMV infection hospitalised at the Department of Paediatric Infectious Diseases in Brno within January 2000-November 2004.

Methods: The diagnosis of congenital HSV, VZV and CMV infection was established partly clinically on the base of typical symptoms of congenitally acquired disease, in a part of infants even from personal anamnesis (mother's disease during pregnancy) and especially verified by isolation of viruses from skin lesion, urine or occasionally from cerebrospinal fluid, detection of VZV, HSV and CMV DNA in the CSF, blood, or surface specimen, and/or by detection of viral antigen and by means of serological examinations.

Results: There were no death in HSV and VZV study group. In the set of 18 newborns with confirmed congenital CMV infection, 11 of them have permanent neurological consequences involving motor of psychomotor problems. Five of them suffer from sensoric affection (4· disturbance up to loss of hearing, 1· strabism). Conclusion: The early diagnosis and management of congenital and perinatal infectious in neonate is important and this will depend on the clinical recognition of the condition. Unfortunately, early diagnosis is problematic. Many of these conditions have non-specific manifestations. As most maternal infections are asymptomatic, repeated serological screening of all susceptibile seronegative women would be required throughout pregnancy. This difficulty exemplified by neonatal herpes infection when early diagnoses and treatment is essential to prevent possible adverse consequences. Early initiation of therapy is of outmost importance every case of herpesviruses infection should be treated as soon as possible. Antiviral therapies have dramatically improved survival rates. Objective: Experimental herpes simplex virus type 1 infection in mice parallels that which occurs in humans. During primary infections, viral replication at the surfaces mucosal is detectable for a 10-to 12-day period. Within 24 h post infection (p.i) HSV infections are common and produce not only a primary infection, but also latent and recurrent infections. Following primary infection of herpes simplex virus type-1, the virus gain access to the termini of sensory neurons, transported in retrograde direction to the neuron cell bodies in sensory ganglia, replicates, spreads to other neurons, and establishes a lifelong latent infection in a portion of the neurons. Method: In this study we used PCR analysis for detection of HSV-1 latency in inoculated BALB/c mice. Thirty five to forty days after intraocular (IO) and intraperitoneal (IP) challenges in different groups, the surviving mice were euthanized, trigeminal ganglia (TG) were harvested, mouse TG was individually homogenized and cell pellet was used to detect viral DNA by PCR analysis .The TG pellet after washing with PBS, were suspended in Tris-EDTA containing .1%SDS and protienase K. After incubation in 55 for 5 h, viral DNA was extracted and PCR performed using two primers of thimidine kinase gene of HSV-1 that generated a 398 bp product. Results: We show that the rate of virus latency in IO infected mice is significantly higher than IP infected mice. Conclusion: Primary HSV infection of any of the 3 branches (ophthalmic, maxillary, mandibular) of cranial nerve V can lead to latent infection of nerve cells in the trigeminal ganglion then, after IO challenge we have more per cent of latency in comparison to IP challenge. Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 Methods: We examined 36 hospitalized patients with suspect HCMV infections (8 children and 28 adults) and 25 blood donors as control group. HCMV pp65 antigen in blood leukocytes were detected and specific IgM and IgG antibodies in sera wera determined by DF and EIA, respectively. The number of blood leukocytes was >2000/ml. Results: Active HCMV infection was diagnosed in22 adults and 6 children (77.7%) by pp65 antigen detection ,while in control group antigenaemia not found. In all cases IgM antibodies were positive in 31 patients (86.1%), IgG antibodies were positive . In 5 children (13.8%) was found seroconversion of IgG .In control group, Igm antibodies were negative, while in 18 of them (72%) igG antibodies were found positive. From the 6 children with active HCMV infection 2 had mononucleosis-like syndrome and 4 acute febrile infection. Adults in respectively from the 22 patients, 6 had mononucleosis-like syndrome, 4 acute febrile infection and 12 undergoing immunosuppressive therapy. The specificity and the sensitivity of antigenaemia assay for diagnosis active infection were 100% and 87.5%, respectively. Conclusions: In this study the pp65 antigen assay seems to be simple, rapid, inexpensive test for early diagnosis of active HCMV infection.

Identification of two major human cytomegalovirus gB genotypes in Taiwan Objectives: Genotypic preference of human cytomegalovirus (HCMV) can be found in a certain geographic area. To determine the predominant CMV genotype(s) in Taiwan, an island located in the southeastern Asia, we performed this study. Methods: We used restriction fragment length polymorphism (RFLP) analysis to determine the subtypes of the HCMV gB gene, which encodes a viral membrane glycoprotein. A total of 101 clinical HCMV isolates collected island-wide were tested to determine their genotypes. Results: We found two major CMV gB genotypes, gB1 (52%) and gB3 (33%), in the paediatric patients. There were 9% of mixed gB types 1 and 3 and 6% of unknown types. None of them belonged to gB types 2 and 4. The reason that results in the significant difference of gB genotypic preference is unclear. Conclusions: Two major HCMV gB genotypes, gB thpe 1 and 3, are found. RFLP may serve as a convenient method for genotypic analysis of CMV clinical isolates.

IgG response to herpes simplex virus type 2 gG and gD recombinant proteins among adults and children Objectives: Recent studies have shown that both HSV-2 glycoprotein D (gD2) and glycoprotein G (gG2) have high potential as diagnostic reagents for the reliable detection of the type-specific IgG. The purpose of this study was to evaluate specific IgG response to HSV-2 gG2 and gD2 among child and adult populations and to investigate the protein-specific pattern of low avidity IgG reactivity.

Methods: A total of 160 serum samples from adults (normal blood donors) and 55 serum samples from children (age range 2-3 years-old) were analysed by type-specific HSV2 IgG enzyme immunoassay (DS-EIA-ANTI-HSV2-IgG, Russia), based on recombinant HSV 2 gG2 (525-578aa) and gD2 (266-394 aa)

proteins. All HSV-2 IgG positive samples were additionally tested for presence of IgG to gD2 (anti gD2) and gG2 (anti gG2) individually. The determination of IgG avidity was performed with 8M urea as a dissociative agent.

Results: 73 out of 160 (45.6%) serum samples from normal blood donors (adults) and 14 out of 55 (25.5%) serum samples from children were positive for HSV2 antibodies by EIA. Protein-specific distribution of IgG activity was significantly different in terms of both IgG level and IgG avidity for two investigated groups (Table 1, Table 2 ).

Conclusion: Specific IgG response to HSV 2 gG2 and to gD2 differs among various age groups. Frequency of low-avidity antibodies in children significantly exceeds frequency of lowavidity antibodies in adults. Furthermore, low-avidity anti gG2 was found predominant in children, and low-avidity anti gD2 in adults.Serologic assay based on the only protein (gG2 or gD2) has a potential for false negative result. It may be especially important for primary HSV2 infection detection.

Qualitative DNA PCR for early detection of symptomatic CMV infection after renal transplant V. Kanapeckiene, R. Valinteliene (Vilnius, LT)

Objectives: One of the most perspective methods in CMV infection diagnostics is detection of viral DNA by the polymerase chain reaction PCR. It is very actual for early differential diagnostics of the arising acute rejection crisis and CMV clinical syndrome after transplantation. Treatment strategy of these clinical syndromes is different: in the crisis of acute rejection, immunosuppressive treatment is intensified, while on the onset of the CMV clinical syndrome-minimised with additional administration of specific antiviral treatment.

Methods: There was carried out a prospective laboratoryclinical study of patients after renal transplantation (duration -3 months). Blood samples for the CMV serological tests were collected once every 10-14 days. In a retrospective way, for the tests by the DNA PCR there were selected 24 kidney recipients with manifesting clinical symptoms over the all first months following transplantation. By the repeated serological tests (ELISA, indirect immunofluorescence), for the patients with manifesting clinical symptoms there was found a diagnostic increase in antibody titres indicating active CMV infection.

The CMV clinical syndrome manifesting in self by fever and other typical clinical signs was determined for 13 persons. In 11 cases the CMV infection was asymptomatic. A positive PCR test was obtained for 12 recipients out of 13 with symptomatic CMV infection and only for 5 out of 11-with asymptomatic infection. A negative PCR test was obtained for 6 patients with the asymptomatic infection and only for 1 with the clinical manifestation of the CMV infection. Thus, using the qualitative PCR method when only a positive or negative result is obtained without estimation of the amount of viral nucleic acids, the symptomatic CMV infection with early onset may be identified in 92.3% of cases (sensitivity of methods), though a positive result in also obtained in 29.4% of cases of the asymptomatic CMV infection. Therefore, specificity of method in as low as 54.4%. Conclusion: The positive prognostic value of the PCR test while seeking to have early detection of the symptomatic CMV infection requiring appropriate treatment is as low as 70.6%. Somewhat more valuable is the negative prognostic value of the PCR test-in the presence of a negative result, in 85.5% of cases there is no clinical manifestation of the CMV infection, and in such cases a specific antiviral treatment is not needed.

Methods: Cases were the patients those admitted to tertiary care hospitals in 2003 with an illness consistent with CCHF and a positive IgM or RT-PCR test result. Two controls per case were selected randomly among those living in the same villages but not experienced a similar illness during the last 3 years. As being potential confounding parameters, age (+10 years) and gender were controlled. A questionnaire was filled by home visits at the region while a blood sample was obtained for serologic screening of specific IgG and IgM antibodies.Statistical analysis was performed by the software NCSS (Number Cruncher Statistical Systems, ver. 2004) . Dichotomous variables were compared by Chi-square or Fisher's Exact Test where applicable. Statistical comparisons were always two tailed.

Results: A total of 62 cases were reachable. Accordingly, 124 controls were selected. Among the risk factors compared only Ôtick exposure before the unset of diseaseÕ was significant (OR, 13.33; CIs, 6.39-27.81). Cases those experienced ticks bites mostly described domestic animals and rural areas out of villages for tick exposure (data not shown). Serologic screening between cases and controls were significant, as well. These comparisons and demographic characteristics, such as age and gender were shown in Table 1 . Discussion: This study demonstrated that tick bite was the single significant risk factor responsible for this outbreak. Animal keeping, buying and selling is widespread in the region (83.3%). Domestic animals could be a source for tick exposure. However, some cases described certain areas out of villages for thick exposure, as well. One important point was that, cases were mostly scattered. Except few examples, they were mostly from different houses. If the live stocks are the source of infected ticks we would found clusters from same houses. The significant difference in the serologic survey between cases and controls supported this view as well. This study, in other words, indicated that rural areas with a heavy tick infestation must be the most likely source of this outbreak. However, more studies in the region are warranted to explore the source of this outbreak and eventually to control it.

High prevalence of Helicobacter pylori infection in Tunisia (414) referred to our hospital laboratory for blood tests were asked to reply to a questionnaire and give a blood sample for a test measuring out the anti-HP antibodies (anti-HP EIA QUANT, Cobas Core II, Roche).234 asymptomatic volunteer blood donors (AVBD) were also included (mean age: 22 yr).

Results: 414 patients were included (52 % male) with mean age of 31 yr (extr: 1-80).94% of these patients were of average social standing. 9.2% were complaining of dyspepsia and 14.2% reported symptoms of dyspepsia in the household.The seroprevalence of HP antibodies was 9% in the young children (1-5 yr) , 40% in the older children (6-15 yr) and 60% in patients between 16 and 20 yr of age. In patients older than 20 yr, seroprevalence was 88% (279/317) comparable to AVBD: 86.3% (202/243). Except for the age, there was no other factor (gender, origin, social standing, dyspeptic symptoms) influencing the HP infection seroprevalence.

Conclusions: Seroprevalence of HP infection is high (79.3%) in our mainly asymptomatic population. Infection increases during childhood and adolescence, reaching 88% in adults. This high prevalence raises the question of whether we should continue to systematically look for HP infection in patients with upper gastroenterological disorders.

A case of acute melioidosis in a traveller Burkkolderia pseudomallei is the causative agent of melioidosis and a potential biological weapon. This anthropozoonosis is endemic in regions of southeast Asia and northern Australia and sporadic in Europe. Bacteraemia in melioidosis is associated with high mortality.We report a case of acute melioidosis in a traveler. A 52-year-old man was admitted to hospital with fever, asthenia and vomiting. He had recently traveled in Bangladesh and Vietnam. He had a documented non-insulino-dependent diabetes. On clinical examination he presented fever and abdominal pain. The diagnostic of malaria was excluded after laboratory investigation. CT abdominal scan showed hepatic and splenic abscesses and the diagnosis of visceral amebiasis was discussed although amebic serology was negative. Of four blood-culture bottles, one aerobic bottle was positive with Gram-negative bacilli identified as Burkholderia cepacia by Vitek 2 (very good identification, T-index 0.66). In regard to the doubtful diagnosis of visceral amebiasis and the unusual susceptibility of the isolate to imipenem, a new identification was made by 16S RNAr sequencing and API20-NE that identified a Burkholderia pseudomallei. An appropriate antimicrobial treatment was then prescribed for a total duration of 12 weeks (ceftazidime i.v and association of trimethoprimsulfamethoxazol/doxycycline). Clinical outcome was favorable.This case emphasizes: 1/the possibility of misidentification of Burkholderia pseudomallei in laboratories that are unfamiliar with this microorganism; 2/the importance of an acute diagnosis of melioidosis to treat correctly this severe disease and to prevent relapses; 3/the necessity to consider the diagnosis of melioidosis in travelers.

Acute gastroenteritis caused by Aeromonas jandaei -importance of identification to the species level E. Durnova, A. Andelova, I. Porazilova, J. Kulhavy, I. Sedlacek (Ostrava, Brno, CZ)

Objectives: Aeromonads have recently been accounted into a new emergent pathogens. They can cause wound infection or septicaemia as well as gastroenteritis. Until now, the epidemiology is not known too much and lately described species still appear. According to our experience the identification of clinical isolates of mesophilic aeromonads only as A. hydrophila, A. caviae, or A. sobria complex is no longer accurate. With proper identification system even so-called rare species can occur in clinical samples. Methods: 160 isolates of aeromonads originated mostly in stool samples. Biochemical identification was done by ENTEROtest24 (Pliva-Lachema), results were assumed by software TNW for oxidase positive fermenting rods. Cellular fatty acid analysis was performed by MIS Sherlock (MIDI, Inc., USA). Results: Genus specific tests such as resistance to vibriostatic agent O/129 (150 lg) and growth in broth culture with addition of NaCl (0%,1%,6%) confirmed, that ENTEROtest24 ensured good distinction of genera Vibrio and Aeromonas. In combination with TNW software ENTEROtest24 was useful for identification of common aeromonads. Nevertheless, due to permanent changes in taxonomy the supplementary conventional biochemical tests are recommended. Routine species identification of Aeromonas isolates by biochemical testing and fatty acid analysis proved the presence of species from complexes A. hydrophila (A. hydrophila, A. bestiarum), A. caviae (A. caviae, A. eucrenophila, A. media) and A. sobria (A. veronii, A. sobria). Beside these common ones we noticed also Aeromonas jandaei isolate from stool sample. Diagnosis of acute gastroenteritis was evident (vomiting, watery dirrhoea. Isolate was identified by ENTEROtest24 as excellent (T index 0.882, ID 91.39%) and comparison of cellular fatty acid profile confirmed the identification. The same major and also minor fatty acids as by A. jandaei CCM 4355T (C16:1w7c alcohol, C16:0N alcohol, C17:0 10 methyl) were presented by our isolate.

Conclusion: There are enough aeromonads in clinical samples and there are also less frequent Aeromonas species occuring in our region. Use of an appropriate identification system can easily warn microbiologist of a rare species. Only proper identification of each isolate will provide us with information, whether all described mesophilic Aeromonas species are involved in diarrhoeal disease.Our project is being supported by IGA of Ministry of Health of the Czech Republic, Id.code: NR/8011-2.

Q-fever febrile illness and pneumonia in a rural town in the Balkans M. Tiholova, G. Kamenov (Sofia, BG)

Objectives: In the spring of 2004 an increase was noted inpatients with acute fever and pneumonia in a small rural town in the Balcans. The diagnosis Q fever was established and the epidemics was limited. 51% of the outbreak cohort had specific antibody titres to phase II C.burnetii antigen >1:64, that confirmed a recent infection. The objectives of our study were to establish the clinical, epidemiological and laboratory parameters of Q fever in this part of Bulgaria and to draw conclusions concerning the early diagnosis, treatment and prevention of the infection in the endemic regions. Methods: To establish the diagnosis of Q-fever all patients had a complete physical examination, epidemiological data were collected. Atypical pneumonia was documented. Seroconvertion was considered in patients demonstrating titres of antibody (IgG > 200 and IgM > 50) to phase II C.burnetii. Results: 51% of seropositive cases presented acute self-limited illness, 39%-atypical pneumonia. Common clinical symptoms were found and appropriate antibiotic regiments indicated.

Conclusions: The apparent reason for the outbreak was the inhalation of infected aerosols. The large number of infected domestic animals in the town may have been the cause. The character of flue-like illnesses and pneumonias during May implies a point source. The early diagnosis of Q fever in risk regions can be helped by epidemiological data on morbidity due to influenza-like illnesses and atypical pneumonia. In such conditions, physicians must treat with appropriate antibiotics before serological confirmation of the diagnosis of Q fever. The public health, veterinary and municipal authorities must work together to educate the population about the basic principles of Q fever prevention which includes restricting contact between people and cattle and improving infection control in the places where animals are bred. Discussion: The apparent reason for the outbreak of atypical pneumonia due to C. burnetii was the inhalation of infected aerosols.The large number of infected domestic animals in the town may have been the cause. The early diagnosis of Q fever in risk regions can be helped by epidemiological data on morbidity due to influenza-like illnesses and atypical pneumonia. In such conditions, physicians must treat with appropriate antibiotics before serological confirmation of the diagnosis of Q fever. Capsular polysaccharides from Neisseria meningitids groups A, C, W135 and Y induce protective bactericidal antibodies in humans, having been used as vaccines for almost two decades.

In contrast, group B capsular polysaccharide vaccines have proven to be essentially nonimmunogenic and for this reason's serogroup B is presently the major cause of meningococcal disease in most developing countries and this, combined with the lack of immunogenicity of the B polysaccharide, has necessitated development of alternative vaccines based upon the outer membrane proteins and lipooligosaccharides. Major surface components, such as the outer membrane porin PorA (class 1 protein), are nowadays considered as potential vaccine candidates. Sergroup B N. menigitidis strains CSBPI, G245 obtained from the Collection of Standard Bacteria Pasteure Institute of Iran for farther investigation the cultivation medium was meningococci medium according to Frantz, with main compotent: L-glutamic acid, L-cystein, glucose and yeast extract. Cell's were cultivated at 36oC for 18h.Outer membrane vesiclecontaining class1(PorA) were prepared and purified according to the Deoxycholate Ultracentrifuge-Differentiation Technique (DUDT) of fermenter grown bacteria. Briefly N. meningitides CSBPI, G245 Was cultivated in Frantz medium supplemented with 0.2% yeast extract dialysate in 40 l fermenter contact-Flow b.v. Bilthoven unit system until early stationary phase and OMV-PorA extract by tris-buffer, EDTA and 0.5% w/v deoxycholate. Purification was done by sequential centrifugation steps at 20,000 g for 30 min. Following ultracentrifugation at 125,000 g for 2 h, the pelleted OMV-PorA were homogenized in PBS. OMV-PorA were ultrasonically treated to disperse the vesicles and were attached to Formvar/Carbon coated Nickel Grids. After preparation step's, the Grids were examined in a Zisse CEM 902A electron microscope. In negative contrast staining and electron microscopy size of OMV-ProA ranged 50-150 nm. Intactness of vesicles in these preparation ranged 70-90% of the vesicle.Protein concentration was measured according to method of lowry et al. The samples were analysed on 10% poly acrylamide gels in the presence of 2% (w/v) SDS. After electrophoresis protein were stained by 0.1% (w/v) coomassie Brilliant Blue staining and the relative amount of OMV-PorA were determined. The results of lowry method was 6 mg protein per 1 ml of sample. When analysed on SDS-PAGE can be separated and their MW can be calculated.

Immunological 300 Pseudomonas aeruginosa strains were isolated from patients admitted in four Tehran hospitals. Using standard O-specific typing sera, they were all grouped in 16 serotypes out of 17 known Pseudomonas aeruginosa. The serotypes were lyophilized and each was given a code according to the Collection of Standard Bacteria Pasteur Institute of Iran (CSBPI) for farther investigations. Among all clinical samples, CSBPI: 16-190 was the most prevalent Pseudomonas aeruginosa serotype which showed a high agglutination titre (4+, 320) against homologous O-specific typing sera. This serotype was selected for extraction of Pseudomonas aeruginosa major outer membrane vesicles (OMP-F). OMP-F vesicles were extracted and purified according to the Deoxycholate Ultracentrifuge-Differentiation Technique. Purity and molecular weight of OMP-F was determined by SDS-PAGE and ability of OMP-F vesicles to induce high titre of antibody in rabbit was shown as a sharp antibody-antigen precipitation line in gel double diffusion test. Passive immunization of mice with anti-rabbit OMP-F antisera, induced a high level of protection when the mice were post-challenged with 2·LD50 of live Pseudomonas aeruginosa CSBPI: . Besides, active immunization of mice with 50 lg of OMP-F, could protect mice against 2·LD50 of live homologous (100% protection) and 15 heterologous native Iranian Pseudomonas aeruginosa serotypes with 50-100% level of protection. OMP-F was Pyrogen-free in rabbit pyrogenic test and did not produce any detectable abnormal toxicity in mice and guinea pigs. Moreover, OMP-F did not show any skin sensitization when 25, 50, 100, 250 and 500 lg were injected intradermally.The above investigations indicate that purified OMP-F of CSBPI: 16-190 can be regarded as a safe protective immunogen in vaccinothrapy against all Pseudomonas aeruginosa immunotype isolated in Iran.

Expression and purification of Herpes simplex virus type 2 glycoprotein D in insect cells using the Baculovirus expression system Objectives: Herpes simplex virus type 2 (HSV-2) infects mucocutaneously and causes genital infection in humans, followed by the establishment of a latent infection in the sensory ganglia and can reactivate for life. A need for effective vaccine remains the preferred strategy for control of HSV-2 infection. Glycoprotein D (gD) of HSV has been focused on as subunit vaccine due to its primary role in inducing cellular and humoral immune responses to a herpes infection. In this study, HSV-2 gD was expressed in insect cells using the Bac-to-Bac Baculovirus Expression System in order to apply as a subunit vaccine in animal models. Methods: The HSV-2 gD of an Iranian isolate was amplified by PCR and cloned into pFastBac plasmid. The recombinant plasmid was transformed into E. coli DH10Bac cells containing bacmid, a baculovirus shuttle vector, for the site-specific transposition of the gD2 gene. The recombinant bacmid was confirmed by PCR, transfected into Sf9 cells, cultured in serumfree Grace's medium at 27°C and monitored daily for observation of cytopathic effects. After 72 h, the baculovirus was harvested from the cell culture medium. The viral stock was amplified by reinfecting insect cells and titrated as pfu. The Sf9 cells were infected with gD2-baculovirus and incubated at 27 ordm;C for various times to express the recombinant protein of interest. The cells were pelleted by centrifugation, lysed and subjected to SDS-PAGE before and after purification. The gels were analysed by western blotting using a monoclonal anti-gD2 antibody.

Results: The gD gene of HSV-2 was cloned after confirming by sequencing. The protein production in insect cells was shown by western blot analysis. The results revealed that Glycosylated and nonglycosylated forms of gD2 were expressed in insect cells as cell-associated proteins by 72 hours post infection.

Conclusion: Baculovirus expression system provides correct folding of recombinant protein as well as disulfide bond formation, oligomerization and other important post-translational modification. Consequently, the expressed protein exhibits the proper biological activity and function. The pure gD2 can be used as a subunit vaccine in animal models.

Seroprevalence of Rubella antibodies among young population in Tehran, Iran H. Soleimanjahi, T. Bamdad, F. Fotouhi Chahooki, M. Roostaee, S.D. Siadat (Tehran, IR)

Objectives: Congenital rubella is a preventable disease, which has largely been controlled through immunization. The aim of this study is to examine the prevalence of antibodies against rubella in Iran, before and after the largest mass campaign for measles and rubella (MR) vaccination of 33,000,000 people with an age rang of 5-25 years-old in 2004.

Methods: A total of 400 serum samples before and after vaccination were collected and assayed using hemagglutination inhibition test (HIT). HIT considered as a reliable index of the immunity level to RV infection. Results: The data revealed that 95% of target population becomes seropositive after vaccination, while 94.25% of them had rubella antibodies before MR vaccination. Conclusion: Rubella is an infectious viral disease, which is endemic in Iran. The more detailed epidemiological studies are still needed to perform a national immunization programme. Based on the results, rubella virus is circulating among population in Iran, so it is recommended to selective vaccination of girls near the age of marriage. Objectives: One of the method of approach for chronic hepatitis B treatment is activation of T-cell immunity. For this purpose therapeutic vaccine containing HBc-ag can be used. Liveattenuated Salmonella are able to deliver both HBc-ag, encoded in prokaryotically expressed plasmid, and HBc-ag, encoded in eukaryotically expressed plasmid. Therefore such mentioned above two types of recombinant Salmonella strains were examined in experiments in mice and cell culture of human monocytes in vitro. Methods: Attenuated cya (lack adenylatecyclase) suppressor (CRP) mutant of Salmonella Enteritidis was used as a vechicle for both HBc-ag encoded in prokaryotically expressed plasmid pKHBc (vector's vaccine), and HBc-ag encoded in eukaryotically expressed plasmid pcDNA3.1pKHBc (DNA-vaccine carried in Salmonella). Mice were inoculated per rectum with suppositories containing 1,000,000 cells of examined Salmonella strains.

Salmonella multiplication in Payer's patches, spleen, liver, lungs, and determination the titre of specific anti -HBc-ag antibodies were examined. Culture of human monocytes in vitro was used for examination of interaction Salmonella strains with phagocytes.

Results: All strains possessed ability to multiply in Payer's patches more then 7 days. In contrast to other Salmonella strains, strain carrying pcDNA3.1pKHBc (DNA-vaccine in Salmonella) did not possessed ability to multiply in parenteral lymphoid tissue. It did not also induce production of specific anti HBC-ag antibodies. Examination of interaction Salmonella strains with culture of human monocytes in vitro was shown that strains free from HBc-ag synthesis possessed ability to multiply in phagocytes. Objectives: The studies aimed at evaluation of anti-S. aureus humoral response in patients treated with autovaccine, prepared from killed S. aureus strains isolated from the patients.

The studies were performed on 15 strains of S. aureus isolated from patients with chronic inflammatory processes in the throat and/or skin and demonstrated to be of staphylococcal aetiology. Surface proteins were isolated by Laemmli buffer and were electrophoretically separated (SDS-PAGE), stained with Coomassie blue or transferred onto nitrocellulose membrane for immunoblotting with patient sera, obtained in two time points, i.e. before and after applying the autovaccine. The antigen/ antibody reactions were visualized employing the alkaline phosphatase method. Protein profiles for individual S. aureus strains and immunobloting results were analysed using GelD-OK software (Syngen Biotech). Results: Using Western-immunoblotting, the IgG lass antibodies were fund to react to S. aureus surface proteins of 14-140 kDa molecular weight while IgM class antibodies reacted with proteins of molecular weight of 25-94 kDa. In studies on the autovaccines IgM antibodies were detected directed toward individual polypeptides in 12 patients while no IgM class antibodies could be detected in the remaining 3 patients. In the subsequent time point of the study (after administration of autovaccine), in 12 patients IgM class immune response was found to resemble that documented before while the remaining, previously negative 3 patients this time carried IgM antibodies specific toward individual polypeptides. As far as IgG class humoral reaction was concerned, the response was comparable in the two time points of the study.

The obtained results suggest that therapeutic effect of S. aureus autovaccine is not related to the respective humoral response.

Clinical Microbiology and Infection, Volume 11, Supplement 2, 2005 and 2003, and PIV-3 infections restricted to march 2003 and January 2004.However,both Ad andPIV-3 did not show any significant correlation with the cilamatic factors.Males were affected more than females, and most infections were associated with lower age groups(less than 1 year). Croup (laryngotracheobronchitis) was only associated withHPIV-3,Bronchiolitis with RSV, bronchopneumonia, pharyngitis and tonsillitis were mostly associated with Ad infections. According to the clinical and radiological findings, RSV infection can be distinguished clinically fromPIV-3 and Ad infections by the higher rates of hypoxemia, retractions, tachypnoea, hyperinflation, and interstitial infiltrates.however.PIV-3and Ad infections cannot be distinguished on the same basis.

Conclusion: This study has demonstrated the prevelance of three othe major respiratory viruses infecting Jordanian children below the age of 2 years, and causing their hospitalzation. The study stated the seasonabilty of repiratoy viruses and their associted illnesses, as well as the major clinical manifestations associated with each virus. The knowledge of local climatic factors together with the characterstic pattern of these viruses may help in the prediction of the beginning and end of epidemics and thus also help in the planninig of preventive programmes.

Tuberculous meningitis in sucklings Results: The were identified in medium 29% new cases in children per year, comparatively with adults (14.8%). From these 23 cases,60% were boys and 26% of the group of age 1-4 months. The onset was incidious in 72,84% , with meningeal syndrome in 91% cases and conscience troubles in 8% cases. The CSF was clear in 95% cases, with high values of proteins and low values of glucose and chlorus. M. tuberculosis was isolated in culture in 28% cases and 14.66% by directly exam. The therapy was then with quadruple association of antituberculous drugs, with favourable evolution 65% cases, 8 deaths being recorded. The factors of bad prognoses still remain: the little age, the hydrocephalus (5 cases), malnutrition (3 cases), and coma from the beginning (2 cases). Conclusion: Tuberculous meningitis still remain a disease with a high incidence and severe evolution in sucklings, despite the progress of diagnostic methods and therapy.

Clinical course of urinary tract infections in early infancy

The majority was virologically stable on HAART (53%); virological response (normalization of liver transaminases and negative HCV RNA) was observed in 11 (65%) patients, and at 12 weeks 15 (88%) patients were HCV RNA negative. Two patients (12%) did not complete therapy up to 12 weeks due to severe depression. At the end of treatment 15 (88%) patients were still HCV RNA negative, and 9 (53%) patients who overcame 48 weeks are always HCV RNA negative. Conclusion: After 24 weeks of acute HCV treatment with peginterferon and ribavirin 15(88%) of treated patients were HCV RNA negative. Peg-interferon and ribavirin show good antiviral efficacy in HIV/HCV co-infected patients

R2190 Infective endocarditis in a group of patients at risk for HIV infection: analysis of 63 episodes

A progressive study of mycobacterium isolations in HIV+ patients

Methods: From 1212 HIV+ patients studied, 156 mycobacterium strains were isolated from various clinical sources. Laboratory testing included Ziehl Neelsen strain, cultures performed classically and automatically by the use of Bactec and MGIT systems, detection of M. tuberculosis rRNA in clinical specimens by the Amplified Mycobacterium Tuberculosis Direct Test AMTD (Gen Probe), identification by the nucleic acid hybridization method of Accuprobe (Gen Probe) and Innolipa V2, (Immunogenetics) and finally, susceptibility testing, by the method of proportion on solid medium Lö wenstein Jensen and in liquid medium by the use of Bactec and MGIT automated systems Innolipa RTF TB (Immunogenetics) for the detection of rpo B gene was also performed

M. tuberculosis was isolated, while in 65 (41.7/%) M. avium. In particular, M. tuberculosis was isolated in 77 (58.8%) men and in 14 (56%) women, while M. avium in 54 (41.2%) men and in 11 (44%) women. M. tuberculosis was mainly isolated from lower respiratory tract, urine and CSF, while M. avium from blood and bone marrow. Among the 91 M. tuberculosis isolations, 87 (95,6%) were susceptible to all antituberculous drugs. Conclusions: Mycobacterium isolation frequency in HIV+ patients is progressively decreasing. M. tuberculosis is predominating in comparison to M. avium. In M. tuberculosis isolations not any significant resistance to antituberculous drugs was detected

Clinico-evolutive aspects of tuberculosis in horizontally HIV-infected patients

Peftidis (Craiova, RO) Objectives: Evaluating the incidence, clinical forms and evolution, of tuberculosis (TB) in horizontally HIV/infected patients

7%) of TB, the diagnosis of HIV infection was established concomitantly with TB diagnosis. Pulmonary forms were: primary -35 cases (51.4%), miliary -six cases (8.8%), infiltrative-ulcerative -seven cases (10.3%), infiltrative-nodulary -five cases (7.4%). Bacteriologic confirmation was found in 10.7% and histopathological in all cases with ganglionary TB

Conclusions: TB remain one of most frequent opportunistic infections in HIV+ patients with advanced immunodepression having a potential severe evolution

Methods: We enrolled 19 patients (CDC stage: 9A, 4B, 6C), 84.2% male, median age 43 years, median time on prior ART 95 months, median CD4 564 cells/ml, 36.8% with undetectable VL and 84% placed on a TDF-containing regimen. Reasons for starting on ATV/RTV were 47.4% hyperlipidemia, 26.3% virological failure and 26.3% toxicities. 10.5% were coinfected (1 HBV, 1 HCV). 4/19 initially received ATV 400 mg daily according to the protocol, changing 6 months later to ATV / RTV. Median time on ATV was 6.5 months. Results: 5/19 patients had mild to moderate bilirubin elevations (2/5 were on IDV) before enrollment. After the initiation of ATV/RTV 15/19 patients (78.9%) experienced grade 3 or 4 hyperbilirubinemia (due to elevation of indirect bilirubin)

Total bilirubin was returned to normal in all four patients after discontinuation of ATV. Conclusions: ATV was safe and well tolerated

No relation between the above side effects and coinfection with HBV and HCV was noted. 1/5 discontinued ATV, mainly due to protocol requirements. ATV seems to be a more potent inhibitor of the uridine diphosphate-glucoronosyl-transferase than IDV. These data in our study revealed a significantly higher percentage of severe hyperbilirubinemia and jaundice from ATV use than previous studies

TR) Objective: To describe the epidemiological and clinical findings of 87 Acquired Immune Deficiency Syndrome (AIDS) patients from Turkey. Methods: All the patients had been followed in Ankara Numune Education and Research Hospital between 1993 and 2004. Results: Seventy-two per cent of the patients were male

GR) Objectives: To evaluate the seroprevalence of hepatitis B surface antigen (HBsAg) in a large multinational group of women at gestational age and to examine the presence of hepatitis B e antigen (HBeAg), antibody to hepatitis B e antigen (anti-HBe) as well as antibody to hepatitis C virus (anti-HCV) in HbsAg(+) ones. Methods: Between

Among women from Albania the seroprevalence of HBsAg was 5.1%, representing the higher rate among the national groups of the study population, followed by 4.2% in Asian women and 1.14% in women from Eastern European countries. The prevalence of HbsAg among African (0.36%) and Greek women (0.29%) was very low. Overall, only 4.45% of HbsAg(+) women were also HbeAg(+) whereas the vast majority of them were HbeAg(-)/antiHbe(+)

Methods: Eighty three samples of sera of forestry workers were tested to detect anti-B.b. and anti-HGE antibodies using ELISA (anti-B.b.) and IFA (anti-HGE) tests.IgG class of antibodies anti-HGE and IgG and IgM classes of antiborrelial antibodies were examined. Results: Presence of anti-HGE IgG antibodies were confirmed in 15 tested serum samples (15/83, 18.1%). Anti-B.b.antibodies were found in 13/83 samples (15.7%)

Serological study confirmed possibility of coinfection with these two pathogens

Patients with L.d.should be tested to detect potential coinfection with HGE

R2225 Brucellosis in municipality of Kërçovë during

Diagnosis of brucellosis was made by following criteria: (a) history of exposure, (b) compatible clinical picture of disease, (c) brucella serum agglutination titre more then 1/160. The method of work is statistical method. R2236 Analysis of anti-flu vaccinations among patients of a family practice: four-year observation I

39 out of 959 patients over the age of 65 i.e 2.76% were vaccinated

this percentage was: 2.42%/4.11%/2.60%. Among patients over the age of 18, in whom anti-flu vaccination is not recommended, percentage of vaccinated patients was: 3.11%/1.85%/3.23%/ 2.27%. Among vaccinated agains the flu at least three times during 2000-2003, we vaccinated: 22% of patients over the age of 50, 59% over the age of 65, 67% of diabetic patients, 90% of those suffering from astma, 32% of patients under the age of 18 and 28% over the age of 18%. Conclusion: Hitherto existing system of anti-flu vaccination promotion is not efficient enough

Abnormal urinalysis results were noted in 65% of infants. Leucocytosis, high C-reactive protein concentration and elevated erythrocyte sedimentation were noted in 45%, 58% and 43%, respectively. 24 infants had abnormal renal ultrasound results (46%). In all cases intravenous treatment with a combination of ampicillin or cephalosporin and aminoglycoside was efficient. Voiding cystourethrogram detected vesicoureteral reflux in 13 infants(24%)

Aim: The aim of our study was to determine the frequency and etiology of infections or colonization of the lower respiratory tract in intubated children in intensive care unit. Material and method: 22 intubated children (15 boys and 7 girls) were studied, aged from 2 months to 16 years-old, treated in I.C.U. during the year 2002. From the study were excluded the chronic cases with frequent re-admissions. The clinical and laboratory infection indexes were evaluated, such as culture of bronchial secretions

Conclusions: 1. Children intubated for more than three days are prone to infection. 2. Colonization of bacteria of the environment may happen the 2nd 24 h of intubation. 3. Children of I.C.U. colonized mainly by Gram-bacteria, S. aureus and Candida spp

The samples were cultured in blood and chocolate agar for 48 h. Identification of the isolates and sensitivity to antibiotics was performed by disk diffusion method following the guidelines of NCCLS. Results: Out of 392 swabs, 285 (73%) were found positive to one or more pathogens. From these in 74 (26%) were isolated 2 bacteria and in 8 (3%) three. The pathogens isolated were 307 (71%) H. influenzae, 38 (8,8%) M. catarrhalis, 24 (5,5%) Candida spp., 15 (3,5%) S. pyogenes (GAS), 8 (1,8%) Neisseriae spp., 4 (0,9%) S. aureus, 1 (0,2%) Streptococcus Group C and 36 (8,3%) Gram-bacteria. From the 38 group A children, 27 (59%) were found positive for H. influenzae, 6 (13%) for M. catarrhalis, 1 (2%) for GAS, 1 (2%) for Candida spp., 1 (2%) for S. aureus and 10 (22%) for Gram-bacteria. 9 swabs (24%) were positive for two pathogens and 1 (5%) for three

Background: The aim of this study was to determine and evaluate pattern changes over the last 9 years regarding the presence of infectious diseases in HIV infected patients during the HAART era. Methods: We retrospectively analysed all infectious episodes in all HIV infected patients from 1996 to 2003. Infectious disease was defined as isolation of a pathogenic bacteria together with consistent clinical findings. No serological data were assessed. We analysed the temporal trend of the most prevalent infections, in search of significant differences to compare their annual prevalence using contingency tables mediating Chi-square analysis Results: 1677 infectious samples in 628 patients were analysed during this period. Significant results are shown in the following Results: 920 patients (p) were evaluated for pancreatic abnormalities in a case-control study including the entire follow-up period of each considered p;128 p with high and prolonged laboratory anomalies were assessed to outline the profile of pancreatic disease before and during the HAART era. Compared with controls, the 334 p (36.3%) who experienced >1 episode of confirmed pancreatic laboratory abnormality had a longer duration of seropositivity, exposure to protease inhibitors, a more frequent immunodeficiency, AIDS diagnosis, liverbiliary disease, and hypertriglyceridemia, while no relation was found with antiretroviral use, and the duration of nucleoside analogue use. Among these 334 p, high and prolonged laboratory alterations eventually associated with signs of organ involvement occurred in 128 p, and were related to the administration of ddI, d4T, 3TC, pentamidine, cotrimoxazole, or antitubercular therapy, substance or alcohol abuse, opportunistic infections, liver-biliary disease, a protease inhibitor-based HAART, and hypertriglyceridemia. However, no difference was noticed between the 32 p with clinical and/or imaging evidence of pancreatic involvement and the remaining 96 asymptomatic p, as to the same risk factors. Although recurrences of enzyme alterations involved >70% of p, in only 33.8% of cases a change of antiretroviral or antimicrobial therapy became needed. An acute but uncomplicated pancreatitis occurred in 7 p of 26 overall symptomatic ones. A 2-4-week gabexate and/or octreotide administration (performed in 59 p of 128), achieved a significant laboratory, clinical, and imaging cure or improvement in 71.2% of p, with a better success rate of combined vs single therapy; a reduced tendency to disease recurrences, and an ameliorated tolerability of antiretrovirals, were also noticed. Conclusion: Epidemiological and pathogenetic studies are needed to assess pancreatic abnormalities especially in the HAART In the case of BDLD transplantation a statistically significative (p = 0.012) increase in the colonization after transplantation was found due to BDLDs (from 25.8% to 61.2%) in contrast with NHBLD transplantation in which NHBLD did not increase the proportion of significant cultures (62.5% in both cases).Conclusions: According to our series NHBLD are statistically less colonized than BDLD. A difference in the proportion of colonization in out hospital NHBLD in the era of prophylactic antibiotherapy could be adventageous in terms of decreasing infections after transplantation, using less prophylactic antibiotics, and avoiding the appearance of antibiotic resistant microorganisms. Objectives: Post-transplant lymphoproliferative diseases (PTLD) is a well recognized complication after HSCT. However, it has been difficult to characterize because the incidence is <1% and clinical presentation is diverse. Incidence and characterisitcs of PTLD after HSCT have not been reported in Korea. This study is aimed to evlauate and characterize PTLD after HSCT in a single institution.

Methods: Medical records of adult patients who underwent allogeneic HSCT in the Catholic HSCT centre from in January 1993-December 2003 were retrospectively analysed. Results: There were 7 patients who had PTLD for 10 years. The mean age was 33 ± 11.6 years, male to female ratio was 6-1. The underlying hematologic diseases were 3 chronic myeloid leukaemia, 2 acute myelocytic leukaemia, 1 myelodysplasic syndrome, 1 severe aplastic anemia. The types of HSCT were 4 of matched sibling donor (MSD) HSCT, 2 of matched unrelated donor (MUD) HSCT, and 1 of haploidenticl HSCT. Five of these patients (71.4%) experienced episodes of acute GVHD and received steroid therapy. PTLD developed at mean 145.3 days (range, 83 244) after HSCT. Clinical symptoms were diverse depending on the involved sites, but cervical lymphadenopathy was frequently observed (5 out of 7). Theincidence of PTLD has been increasing: no proven cases before 1996, 1 case in 1996, 3 in 2001, 1 in 2002, 2 in 2003 . The pathology review showed that 6 cases had B-cell origin PTLD and one patient had T-cell origin, who had haploidentical HSCT and rapidly progressed to death. In situ hybridization for EBER was positve in 4 cases. Treatments of PTLD were 1 acyclovir therapy, 2 reduction of immunosuppressants plus acyclovir therapy, 2 reduction of immunosuppressants plus donor lymphocyte infusion (DLI), 2 reduction of immunosuppressants plus acyclovir therapy plus DLI. Five of seven patients (71.4%) reponded to the treatment. Three patients died, and 2 of them died patially or directly due to PTLD, which developed in 2003 in these cases. Conclusion: The incidence of PTLD is low in allogeneic HSCT patients, but has been increasing recently probably because the rate of HLA mismatched HSCT or MUD HSCT, which needs more profound immunosuppression, has increased. Therefore, we should be careful of development of PTLD in patients after HSCT, who have risks of PTLD.

The prevelance of respiratory viruses in hospitalised children in Amman, Jordan Objectives: To detemine the prevelance, seasonal distribution and to draw out the clinical signs and symptoms associated with the following respiratory viruses:Respiratory syncytial virus(RSV),Human parainfluenza viruses (PIV) type 1,2 and 3,Influenza viruses A&B and Adenoviruses(Ad).Methods: A total of 200 nasopharyngeal aspirates were obtained from hospitalized children (below 2 years old), admit-ted to Islamic hospital in Amman-Jordan, between september 2002 and March 2004. These specimens were analysed for the presence of some respiratory viruses using the direct and indirect immunoflouresence techniques. Results: 27% of enrolled patients have proved viral respiratory infection. The etiological agents associated with these infections were RSV(12%), Ad.(11.5%) and PIV-3(3%). Seasonal distribution of respiratory viruses and their correlation with the climatic factors (temperature, rainfall, and humidity) were determined. The peak of RSV incidince was during February 2003 and January 2004 and showed a significant correlation with the climatic factors. Ad infections peaked during December of2002