Community-acquired Clostridium difficile infections in emergency departments.

OBJECTIVE: Clostridium difficile infection (CDI) has become an emerging infectious disease, especially in community settings. Little data is available on its frequency and characteristics in France. We aimed to describe CDI case patients consulting at the emergency department and to compare community-acquired and nosocomial CDIs. PATIENTS AND METHODS: We conducted a multicenter retrospective study over a three-year period of community-acquired and nosocomial CDI case patients seen at the emergency department and compared their characteristics. RESULTS: A total of 2055 patients consulted for diarrhea during the study period and had a stool culture performed: 66 (3.2%) presented with a CDI, of which 28 were community-acquired and 26 were nosocomial. Community-acquired CDI patients had a mean age of 57.7years (18-91), with a sex-ratio of 0.65. At least one risk factor was observed in 24 patients (85.7%), of whom 22 (78.6%) had been prescribed a previous antimicrobial treatment. Diabetes mellitus and renal failure were more frequently observed in patients presenting with nosocomial CDI. They required fluid replacement and needed be to re-hospitalized more often than community-acquired patients. CONCLUSION: Community-acquired CDIs in the emergency department account for approximately 1.4% of patients presenting with diarrhea. One risk factor is present in 85.7% of cases. In our study, their presentation and outcome seemed less severe than nosocomial CDIs.

Development of antileishmanial lipid nanocomplexes.

Visceral leishmaniasis is a life-threatening disease that affects nearly a million people every year. The emergence of Leishmania strains resistant to existing drugs complicates its treatment. The purpose of this study was to develop a new lipid formulation based on nanocochleates combining two active drugs: Amphotericin B (AmB) and Miltefosine (HePC). Nanocochleates composed of dioleoylphosphatidylserine (DOPS) and Cholesterol (Cho) and Ca(2+), in which HePC and AmB were incorporated, were prepared. Properties such as particle size, zeta potential, drug payload, in-vitro drug release and storage stability were investigated. Moreover, in-vitro stability in gastrointestinal fluid was performed in view of an oral administration. AmB-HePC-loaded nanocochleates with a mean particle size of 250 ± 2 nm were obtained. The particles displayed a narrow size distribution and a drug payload of 29.9 ± 0.5 mg/g for AmB, and 14.0 ± 0.9 mg/g for HePC. Drug release occurred preferentially in intestinal medium containing bile salts. Therefore, AmB-HePC-loaded nanocochleates could be a promising oral delivery system for the treatment of visceral leishmaniasis.

Methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to glycopeptides (GISA) in 63 French general hospitals.

Clinical isolates of methicillin-resistant Staphylococcus aureus (n = 1070) collected from 63 French general hospitals during June 2000 (n = 1070) were screened initially for reduced susceptibility to glycopeptides (GISA) on brain-heart infusion agar containing teicoplanin 6 mg/L. Glycopeptide MICs were determined for the 145 isolates that grew on the screening plates. Of the 1070 isolates, 1.4% were GISA on Mueller-Hinton agar, and 2.9% by Etest with a high inoculum, while 0.7% and 2.9% were GISA by vancomycin and teicoplanin population analysis profiles, respectively. Most isolates were resistant to gentamicin and rifampicin or fosfomycin or fusidic acid, as determined by disk diffusion. Pulsed-field gel electrophoresis of the 31 GISA isolates identified four clones, with dissemination of one predominant clone. In these French hospitals there was a low incidence of GISA and hetero-GISA.

Polymeric carriers for amphotericin B: in vitro activity, toxicity and therapeutic efficacy against systemic candidiasis in neutropenic mice.

OBJECTIVE: To study the toxicity and activity of two new amphotericin B formulations: poly(epsilon-caprolactone) nanospheres coated with poloxamer 188 (AmB-NP) and mixed micelles with the same surfactant (AmB-MM). MATERIALS AND METHODS: The toxicity of these formulations was evaluated in erythrocytes, J774.2 macrophages and LLCPK1 renal cells, as well as in mice. Activity was determined in clinical isolates and in neutropenic mice. Mice were made neutropenic with 5-fluorouracil, infected with Candida albicans and treated with the antifungal formulations for three consecutive days. AmB association in cells and accumulation in kidneys and liver of animals was quantified by HPLC. RESULTS: Both formulations decreased between 8- and 10-fold the MIC of the polyene against clinical isolates of C. albicans. However, their activity was lower than or equal to that of AmB-deoxycholate when it was assessed against C. albicans-infected macrophages. When given as a single intravenous dose in mice, AmB-MM and AmB-NP had an LD50 of 9.8 and 18.6 mg/kg, respectively, compared with 4 mg/kg for AmB-deoxycholate. Comparison of residual infection burdens in the liver and kidneys showed that AmB-deoxycholate (0.5 mg/kg) was more effective and faster in eradicating yeast cells than polymeric formulations. This fact can be related to a lower AmB accumulation inside macrophages and in liver and kidneys (about 1.5 mg drug/g tissue) of mice, compared with those detected for AmB-deoxycholate (4 mg drug/g). Overall, the efficacy of these formulations at 2 mg/kg was equal to that of AmB-deoxycholate at 0.5 mg/kg. CONCLUSIONS: AmB-MM and AmB-NP decreased the in vivo antifungal activity of AmB, and higher concentrations were therefore necessary to obtain a similar therapeutic effect. However, these higher concentrations were achievable owing to the reduced toxicity of these formulations.

Combined hydroxypropyl-beta-cyclodextrin and poly(alkylcyanoacrylate) nanoparticles intended for oral administration of saquinavir.

The aim of this study was to prepare and characterize an hydroxypropyl-beta-cyclodextrin-saquinavir inclusion complex with the purpose of incorporating this complex into poly(alkylcyanoacrylate) nanoparticles in order to increase the drug loading. Hydroxypropyl-beta-cyclodextrin-saquinavir complex was characterized by thermal (differential scanning calorimetry), crystallographic (X-ray diffractography) and spectroscopic methods (circular dichroism, H1-NMR). Nanoparticles were prepared by polymerization of alkylcyanoacrylate monomers (isobutyl- and isohexylcyanoacrylate) in a water solution of the complex and further characterized. The apparent solubility of saquinavir was increased 400-fold at pH 7.0 in presence of hydroxypropyl-beta-cyclodextrin owing to the formation of a drug-cyclodextrin complex as demonstrated mainly by 1H NMR and confirmed by other techniques. Saquinavir-loaded nanoparticles could be easily prepared in the presence of a drug-cyclodextrin complex. It was found that large amounts of cyclodextrins remained associated with the particles, resulting in a 20-fold increase in saquinavir loading compared to nanoparticles prepared in the absence of cyclodextrins. This study has shown that the loading in saquinavir of poly(alkylcyanoacrylate) nanospheres could be dramatically improved by simultaneously increasing the apparent solubility of the drug in the preparation medium and the amount of cyclodextrin associated with the particles, making these nanospheres a promising system for oral application.

[Primary Neisseria meningitidis arthritis of the knee without meningitis: contribution of synovial fluid culture in blood-culture vial]. / Monoarthrite du genou à Neisseria meningitidis sans méningite: apport de la culture du liquide articulaire en flacon d'hémoculture.

INTRODUCTION: Primary meningococcal arthritis is a rare form of meningococcal disease. It occurs as an isolated acute purulent arthritis without meningitis, and presence of Neisseria meningitidis in articular fluid. We report a new case of typical primary meningococcal arthritis. EXEGESIS: A previously healthy 23-year-old female patient was admitted for purpuric lesions of the legs. At admission, conscience was normal and symptoms of meningitis were absent. During the 2nd day of hospitalisation, a warm and painful effusion in the right knee appeared. Aspiration from the right knee yielded a purulent fluid. N. meningitidis was isolated from a blood-culture vial inoculated with the synovial fluid, while blood cultures remained sterile. Anti-biotherapy was initiated as soon as microbiological diagnosis was established. The patient was symptom-free 1 month later. CONCLUSION: We emphasize the fact that agar cultures of the synovial fluid remained sterile, while N. meningitidis grew in a blood-culture vial. We suggest that diagnosis of primary meningococcal arthritis may be underestimated when inappropriate culture media are used.

Lymph node infection by Trichomonas tenax: report of a case with co-infection by Mycobacterium tuberculosis.

In an 82-year-old woman, presenting with fever and asthenia, cervical adenopathy was noted. Clinical and radiological investigations were fruitless. Laboratory examinations detected a refractory anemia. The lymph node was excised and showed numerous trichomonads on touch preparations. Histologically, the node showed caseous necrosis and macrophagic reaction. Diagnosis of lymph node infection by Trichomonas tenax was made. Three weeks later, culture of the node showed Mycobacterium tuberculosis and let us conclude co-infection. T tenax is usually regarded as a harmless saprophyte of the oral cavity. This exceptional observation shows for the first time an invasive potential of T tenax. It raises questions about links with tuberculosis and refractory anemia.

N-Methyl-N-D-fructosyl amphotericin B methyl ester (MF-AME), a novel antifungal agent of low toxicity: monomer/micelle control over selective toxicity.

Rational chemical modification of amphotericin B (AMB) led to the synthesis of sterically hindered AMB derivatives. The selected optimal compound, N-methyl-N-D-fructosyl amphotericin B methyl ester (MF-AME) retains the broad spectrum of antifungal activity of the parent antibiotic, and exhibits a two orders of magnitude lower toxicity in vivo and in vitro against mammalian cells. Comparative studies of MF-AME and AMB comprising the determination of the spectroscopic properties of monomeric and self-associated forms of the antibiotics, the investigation of the influence of self-association on toxicity to human red blood cells, and of the antibiotic-sterol interaction were performed. On the basis of the results obtained it can be assumed that the improvement of the selective toxicity of MF-AME could in part be a consequence of the diminished concentration of water soluble oligomers in aqueous medium, and the better ability to differentiate between cholesterol and ergosterol.

Probable transmission of HIV from an orthopedic surgeon to a patient in France.

BACKGROUND: Transmission of HIV from infected health care workers to patients has been documented in only one cluster involving 6 patients of a dentist in Florida. In October 1995, the French Ministry of Health offered HIV testing to patients who had been operated on by an orthopedic surgeon in whom AIDS was recently diagnosed. OBJECTIVE: To determine whether the surgeon transmitted HIV to patients during operations. DESIGN: Epidemiologic investigation. SETTING: The practice of an orthopedic surgeon in a French public hospital. PARTICIPANTS: 1 surgeon and 983 of his former patients. MEASUREMENTS: 3004 patients who had undergone invasive procedures were contacted by mail for counseling and HIV testing. One HIV-positive patient was interviewed, and DNA sequence analysis was performed to compare the genetic relation of the patient's and the surgeon's viruses. Infection-control precautions and the surgeon's practices were assessed. RESULTS: Of 983 patients in whom serologic status was ascertained, 982 were HIV negative and 1 was HIV positive. The HIV-positive patient, a woman born in 1925, tested negative for HIV before placement of a total hip prosthesis with bone graft (a prolonged operation) performed by the surgeon in 1992. She had no identified risk for HIV exposure. Molecular analysis indicated that the viral sequences obtained from the surgeon and the HIV-infected woman were closely related. Infection-control precautions were in accordance with recommendations, but blood contact between the surgeon and his patients occurred commonly during surgical procedures. CONCLUSIONS: An HIV-infected surgeon may have transmitted HIV to one of his patients during surgery.

In-vivo therapeutic efficacy in experimental murine mycoses of a new formulation of deoxycholate-amphotericin B obtained by mild heating.

Heat-induced 'superaggregation' of deoxycholate-amphotericin B (AmB-DOC, Fungizone) was shown previously to reduce the in-vitro toxicity of this antifungal agent. We compared AmB-DOC with the formulation obtained by heating the commercial form (Fungizone, Bristol Myers Squibb, Paris, France) for 20 min at 70 degrees C, in the treatment of murine infections. An improvement of antifungal activity was obtained with heated AmB-DOC formulations due to a lower toxicity which allowed the administration of higher drug doses than those achievable with the commercial preparation. Single intravenous injections of heated AmB-DOC solutions were demonstrated to be two-fold less toxic than unheated ones to healthy mice. For mice infected with Candida albicans, the maximum tolerated dose was higher with heated than with unheated AmB-DOC solutions. In the model of murine candidiasis, following a single dose of heated AmB-DOC 0.5 mg/kg, 85% of mice survived for 3 weeks, whereas at this dose the immediate toxicity of the standard formulation in infected mice restricted the therapeutic efficacy to 25% survival. Both formulations were equally effective in increasing the survival time for murine cryptococcal pneumonia and meningoencephalitis. Injection of heated AmB-DOC solutions at a dose two-fold higher than the maximal tolerated dose observed with the unheated preparation (1.2 mg/kg) increased the survival time by a factor of 1.4 in cryptococcal meningoencephalitis. These results indicate that mild heat treatment of AmB-DOC solutions could provide a simple and economical method to improve the therapeutic index of this antifungal agent by reducing its toxicity on mammalian cells.

Heat-induced superaggregation of amphotericin B reduces its in vitro toxicity: a new way to improve its therapeutic index.

Superaggregation of amphotericin B (AmB) was previously shown to occur upon heating of solutions at 70 degrees C. In the present study, we demonstrate that heat pretreatment of Fungizone (deoxycholate salt of AmB [AmB-DOC]) solutions induces a drastic decrease in the in vitro toxicity of this antibiotic. Heated AmB-DOC colloidal solutions, which mainly contained superaggregated and monomeric forms of the antibiotic, were strongly less hemolytic than unheated solutions (aggregates and monomers). Thermal pretreatment of AmB-DOC solutions also reduced the toxicity to the cell line HT29, as deduced from two simultaneous cell viability assays (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release). These heated colloidal solutions were only slightly less efficient than the unheated ones at inhibiting the growth of Candida albicans cells in vitro. Such results suggest that mild heat treatment of AmB-DOC solutions could provide a new and simple solution for improving the therapeutic index of this antifungal agent by reducing its toxicity to mammalian cells.

Physico-chemical properties of the heat-induced 'superaggregates' of amphotericin B.

The aggregation state of amphotericin B (AmB) was previously reported to modulate its therapeutic efficiency. As a preliminary study to test the biological effects of 'superaggregates' generated by heat treatment, we present spectroscopic data related to their formation in aqueous solutions. Drastic changes in the AmB aggregation state in water were shown to occur on heating at 50-60 degrees C. The concentration of the aggregates formed at high (A(t)) or room (A) temperature, and the concentration of the monomeric form (M) of AmB were calculated by processing absorption data. The thermally induced conversion from A to A(t) depends on the AmB concentration. Rayleigh scattering measurements suggest that the A(t) aggregates are larger than the A aggregates. At room temperature, the condensation rate of A with M-leading to the 'superaggregated' form A(t)-was slower and depended on the concentration of M. The superaggregated species A(t) was shown to be the most chemically stable species. Physico-chemical properties of these superaggregates are discussed as a potential new solution to improve the therapeutic efficacy of AmB.

Release of amphotericin B from delivery systems and its action against fungal and mammalian cells.

Spectroscopic studies of four amphotericin B (AmB) lipid preparations--small negatively charged unilamellar vesicles "AmBisome", positively charged oligolamellar liposomes "Ampholiposomes", AmB Lipid Complex "L-AmpB33"--and AmB association with gamma cyclodextrin demonstrated that the composition of drug delivery system directly influences AmB organization. The aggregation state and release in external medium of AmB was monitored by circular dichroism and UV-visible absorption. AmB short-term activity against Candida albicans (K+ leakage) was found to be correlated with the amount of free AmB released from lipid preparations. These data seem to indicate that lipid composition influences anti-Candida albicans activity, by modulation of AmB binding to lipids.

Investigation of hospital-acquired infections due to Alcaligenes denitrificans subsp. xylosoxydans by DNA restriction fragment length polymorphism.

We demonstrate that DNA restriction fragment length polymorphism determined by pulsed-field gel electrophoresis is very useful in the investigation of the epidemiology of hospital-acquired infections caused by Alcaligenes denitrificans subsp. xylosoxydans. This approach showed that hospital-acquired infections caused by this opportunistic pathogen over a 6-month period in 10 patients hospitalized in an intensive care unit and a surgical unit were not a true outbreak. In addition, this molecular typing method established that the respiratory therapy equipment was the source of the contamination of two patients.

In vitro renal toxicity and in vivo therapeutic efficacy in experimental murine cryptococcosis of amphotericin B (Fungizone) associated with Intralipid.

We compared the experimental toxicities and activities of deoxycholate amphotericin B (d-AmB) dissolved in glucose (Dd-AmB) or mixed with 20% Intralipid (ILd-AmB). In vitro, ILd-AmB against renal tubular cells in primary culture. In vivo, the toxicities and activities of Dd-AmB and ILd-AmB were studied in DBA2 mice with cryptococcosis. The maximum tolerated dose of intravenously administered d-AmB, i.e., the dose that induced less than 15% mortality because of toxicity, was 1.7 to 2.5 times higher when it was administered as ILd-AmB than when it was administered as Dd-AmB. Both treatments given intravenously at the same dose were equivalent for improving the survival of mice and reducing CFU counts in infected tissue, but at maximum tolerated doses, ILd-AmB (2 mg/kg of body weight) was more effective than Dd-AmB (0.8 to 1.2 mg/kg). AmB concentrations in spleen, liver, lung, and kidney were measured by high-pressure liquid chromatography 4 and 24 h after a single injection of 1.2 mg of Dd-AmB per kg, 1.2 mg of ILd-AmB per kg, or 2 mg of ILd-AmB per kg. In a given organ, AmB levels were similar after administration of 1.2 mg of Dd-AmB or ILd-AmB per kg but were significantly higher after administration of 2 mg of ILd-AmB per kg. The lower level of toxicity of ILd-AmB might be explained by circular dichroism experiments, showing that ILd-AmB contained 10-fold less soluble oligomeric AmB, which is believed to be the toxic form of the drug, than Dd-AmB. We conclude that ILd-AmB is as efficient as Dd-AmB and is better tolerated than Dd-AmB in mice with experimental cryptococcosis. By allowing higher doses of AmB to be infused, Intralipid enhances AmB concentrations in infected sites, and thus the therapeutic activity of the drug.

Quantitative structure-activity relationships in amphotericin B derivatives.

The quantitative structure-activity relationships studies of amphotericin B and its 16 semisynthetic derivatives obtained by modification at carboxyl and amino groups have been done. The results of five biological tests were subjected to principal component analysis, a numerical method useful in the investigation of large sets of data. For some compounds, also, interaction with lipidic vesicles was investigated by spectroscopic methods. The results obtained indicate that: (i) The presence of positively charged nitrogen atom (protonable or bearing fixed charge) is indispensable for biological activity and antibiotic-sterol interaction; (ii) The lack of free carboxyl group in the molecule favours the differentiation between cholesterol and ergosterol containing cells.

Inhibition by Cu2+ of amphotericin B induced lysis of erythrocytes.

Amphotericin B-induced lysis of erythrocytes is diminished in the presence of Cu2+, but the prelytic amphotericin B-induced K+ leakage is unaffected. These results and the weak binding of Cu2+ to amphotericin B, demonstrated by circular dichroism and EPR studies, are consistent with the view that Cu2+ protects erythrocytes by increasing their resistance to lysis.

Effect of surface curvature on the interaction of single lamellar phospholipid vesicles with aromatic and nonaromatic heptaene antibiotics (vacidin A and amphotericin B).

The interactions of unilamellar lipid vesicles with vacidin A, an aromatic heptaene antibiotic, and with amphotericin B, a nonaromatic heptaene antibiotic were compared. Uptake of both antibiotics, monitored by circular dichroism, was found to be faster with small vesicles than with large ones. By combining permeability measurements (Gary-Bobo and Cybulska, J. Antibiotics 35, 1068 (1982)) and circular dichroism spectra, we found that for vacidin A, the same permeability inducing species is formed regardless of vesicles size. However, at a given concentration of antibiotic, less of the permeability inducing species is formed in the presence of small vesicles than in the presence of large vesicles. This may account for the differences between small and large vesicles in antibiotics-induced permeability. For amphotericin B, the permeability inducing species formed in the presence of small vesicles differs from that formed in the presence of large vesicles.