Stefan Zimmerli

Clinical Implications of Mycobacterium kansasii Species Heterogeneity: Swiss National Survey

ABSTRACT Several subtypes of Mycobacterium kansasii have been described, but their respective pathogenic roles are not clear. This study investigated the distribution of subtypes and the pathogenicity of M. kansasii strains (n = 191) isolated in Switzerland between 1991 and 1997. Demographic, clinical, and microbiological information was recorded from clinical files. Patients were classified as having an infection according to the criteria of the American Thoracic Society. Subtypes were defined by PCR-restriction enzyme analysis of the hsp65 gene. Subtype 1 comprised 67% of the isolates (n = 128), while subtypes 2 and 3 comprised 21% (n = 40) and 8% (n = 15), respectively. Other subtypes (subtypes 4 and 6 and a new subtype, 7) were recovered from only 4% of patients (n = 8). M. kansasii subtype 1 was considered pathogenic in 81% of patients, while M. kansasii subtype 2 was considered pathogenic in 67% of patients and other subtypes were considered pathogenic in 6% of patients. The majority of patients with M. kansasii subtype 2 were immunocompromised due to the use of corticosteroids (21% of patients) or coinfection with HIV (62.5% of patients). Subtyping M. kansasii may improve clinical management by distinguishing pathogenic from nonpathogenic subtypes.

Phagocytic Uptake of Encephalitozoon cuniculi by Nonprofessional Phagocytes

ABSTRACT Encephalitozoon cuniculi is an obligate intracellular, spore-forming parasite belonging to the microsporidia that can cause disseminated infection in immunocompromised persons. E. cuniculi spores infect host cells by germination, i.e., by explosively everting the polar filament, through which the spore contents (sporoplasms) are subsequently injected into the cytoplasm. In addition, we observed intracellular, nongerminated spores in various nonprofessional phagocytes. In MRC5 cells, the number of internalized spores was approximately 10-fold higher than the number of injected sporoplasms. Compared to the rate of uptake by human monocyte-derived macrophages, internalization rates by A549 cells, MRC5 cells, and 293 cells were 0.6, 4.4, and 22.2%, respectively. The mechanism of uptake was studied in MRC5 cells. Killed spores were internalized at the same rate as live spores, indicating that nongerminated parasites do not actively participate in cell entry. Cytochalasin D inhibited uptake of spores by 95%, demonstrating an actin-dependent process. By electron and epifluorescence microscopy, intracellular spores were found in a tightly fitting membrane-bound compartment. The vacuole containing the spores was positive for the lysosomal membrane protein LAMP-1 and colocalized with the late endosomal-lysosomal content marker rhodamine dextran. Our results show that, in addition to the unique way in which microsporidia infect cells, E. cuniculi spores enter nonprofessional phagocytes by phagocytosis and traffic into a late endosomal-lysosomal compartment.

Candida species distribution and antifungal susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing and new vs. old Clinical and Laboratory Standards Institute clinical breakpoints: a 6-year prospective candidaemia survey from the fungal infection network of Switzerland

We analyzed the species distribution of Candida blood isolates (CBIs), prospectively collected between 2004 and 2009 within FUNGINOS, and compared their antifungal susceptibility according to clinical breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2013, and the Clinical and Laboratory Standards Institute (CLSI) in 2008 (old CLSI breakpoints) and 2012 (new CLSI breakpoints). CBIs were tested for susceptiblity to fluconazole, voriconazole and caspofungin by microtitre broth dilution (Sensititre® YeastOne™ test panel). Of 1090 CBIs, 675 (61.9%) were C. albicans, 191 (17.5%) C. glabrata, 64 (5.9%) C. tropicalis, 59 (5.4%) C. parapsilosis, 33 (3%) C. dubliniensis, 22 (2%) C. krusei and 46 (4.2%) rare Candida species. Independently of the breakpoints applied, C. albicans was almost uniformly (>98%) susceptible to all three antifungal agents. In contrast, the proportions of fluconazole- and voriconazole-susceptible C. tropicalis and F-susceptible C. parapsilosis were lower according to EUCAST/new CLSI breakpoints than to the old CLSI breakpoints. For caspofungin, non-susceptibility occurred mainly in C. krusei (63.3%) and C. glabrata (9.4%). Nine isolates (five C. tropicalis, three C. albicans and one C. parapsilosis) were cross-resistant to azoles according to EUCAST breakpoints, compared with three isolates (two C. albicans and one C. tropicalis) according to new and two (2 C. albicans) according to old CLSI breakpoints. Four species (C. albicans, C. glabrata, C. tropicalis and C. parapsilosis) represented >90% of all CBIs. In vitro resistance to fluconazole, voriconazole and caspofungin was rare among C. albicans, but an increase of non-susceptibile isolates was observed among C. tropicalis/C. parapsilosis for the azoles and C. glabrata/C. krusei for caspofungin according to EUCAST and new CLSI breakpoints compared with old CLSI breakpoints.

Clinical Characteristics Associated with Isolation of Small-Colony Variants of Staphylococcus aureus and Pseudomonas aeruginosa from Respiratory Secretions of Patients with Cystic Fibrosis

ABSTRACT During a 3-month period, small-colony variant phenotypes of both Staphylococcus aureus and Pseudomonas aeruginosa were isolated from respiratory secretions of 8.2% and 9.2%, respectively, of 98 patients with cystic fibrosis, particularly those with advanced pulmonary disease and prolonged antibiotic exposure.

Ocular Distribution of Intravenously Administered Lipid Formulations of Amphotericin B in a Rabbit Model

ABSTRACT Little is known about the ocular penetration of amphotericin B (AMB) and its lipid formulations, the current drug of choice in fungal endophthalmitis. The ocular distribution of AMB lipid complex (ABLC), liposomal AMB (L-AMB), and AMB deoxycholate (D-AMB) was studied in a rabbit model. D-AMB (1 mg/kg of body weight/day), ABLC (5 mg/kg/day), or L-AMB (5 mg/kg/day) was given intravenously to rabbits as a single dose or as repeated daily doses on 7 consecutive days after induction of unilateral uveitis by intravitreal injection of endotoxin. AMB concentrations in aqueous humor, vitreous humor, and plasma were determined by high-pressure liquid chromatography 16 h after administration of a single dose or 24 h after the last of seven doses. After single-dose administration, L-AMB achieved at least eightfold-higher AMB concentrations in the aqueous of inflamed eyes than ABLC or D-AMB (1.21 ± 0.58 μg/ml versus 0.14 ± 0.04 and 0.11 ± 0.09 μg/ml, respectively). At that time point no drug was detectable in the vitreous. After 7 days of treatment, the concentration of AMB in the vitreous was higher after treatment with L-AMB (0.47 ± 0.21 μg/ml) than after treatment with ABLC (0.27 ± 0.18 μg/ml) and D-AMB (0.16 ± 0.04 μg/ml). Similarly, AMB concentration in the aqueous was higher after repeated doses of L-AMB (0.73 ± 0.43 μg/ml) than after repeated doses of ABLC (0.03 ± 0.02 μg/ml) or D-AMB (0.13 ± 0.06 μg/ml). No AMB was detected in noninflamed eyes. Following systemic administration, AMB distribution to the eye is inflammation dependent and occurs sequentially, first to the aqueous and then to the vitreous. Compared to D-AMB and ABLC, L-AMB reaches higher drug concentrations in both ocular compartments.

Topical Caspofungin for Treatment of Keratitis Caused by Candida albicans in a Rabbit Model

ABSTRACT Candida albicans is the most frequent cause of fungal keratitis in temperate regions. Caspofungin has potent activity against Candida spp. in a variety of clinical settings. Little is known, however, about its activity against fungal keratitis. We compared the efficacy of topical caspofungin with that of topical amphotericin B (AMB) in a rabbit model of experimental keratomycosis. Keratitis was induced with a standardized inoculum of Candida albicans (SC 5314) placed on the debrided cornea. Twenty-four hours after infection, animals were randomly assigned to treatment with 0.15% caspofungin, 0.5% caspofungin, 0.15% AMB, and a saline control (n = 12 rabbits in each group). For the first 12 h, treatment was repeated every 30 min and, after a 12-h pause, was resumed at hourly intervals for another 12 h. The animals were examined and killed 12 h after administration of the last dose. Treatment effects were evaluated by clinical assessment, fungal culture, and histopathology. Drug treatment significantly reduced corneal fungal recovery from 3.78 log10 CFU in saline-treated animals to 2.97, 1.76, and 1.18 log10 CFU in animals treated with 0.15% caspofungin, 0.5% caspofungin, and 0.15% AMB, respectively. By histopathology, the mean hyphal density was significantly lower in the corneas of treated animals than in those of the controls; there was no difference in hyphal densities between the different treatment groups. The depth of corneal invasion was not significantly reduced by the antifungal treatments. By clinical assessment, keratitis progressed in animals treated with saline, whereas disease progression was inhibited by all drug treatment regimens. In our rabbit model, 0.5% caspofungin was as effective as 0.15% AMB for the topical treatment of Candida keratitis. The potential clinical efficacy of caspofungin awaits further investigation.

Treatment of postkeratitis fusarium endophthalmitis with amphotericin B lipid complex.

Purpose. The authors report the first case of Fusarium solani keratitis that progressed to fungal endophthalmitis and was successfully treated with amphotericin B lipid complex (ABLC). Method. The case of a 34-year-old immunocompetent woman who developed a contact lens-related F. solani keratitis requiring emergency penetrating keratoplasty (PKP) was analyzed. The immunocompetent patient developed fungal endophthalmitis (anterior chamber tap positive for F. solani three months after PKP) and was eventually treated with ABLC. Results. Systemic amphotericin B (total, 0.42 g) and ketoconazole in addition to topical natamycin and amphotericin did not prove to be effective in eradicating the mycosis in the anterior chamber. Under ABLC treatment (total, 8.79 g), the anterior chamber inflammation resolved completely. No recurrence was observed during an 11-month follow-up after treatment was discontinued. Conclusion. ABLC proved to be effective in treating F. solani endophthalmitis. It is an important addition to the ophthalmic armamentarium, and appeared to be a better therapeutic agent than standard amphotericin B in this patient.

Emergence of Blastoschizomyces capitatus yeast infections, Central Europe.

We report 5 cases of disseminated infection caused by Blastoschizomyces capitatus yeast in central Switzerland. The emergence of this yeast in an area in which it is not known to be endemic should alert clinicians caring for immunocompromised patients outside the Mediterranean region to consider infections caused by unfamiliar fungal pathogens.

Disseminated Tuberculosis Following Total Knee Arthroplasty in an HIV Patient

Skeletal tuberculosis is now uncommon in developed countries. In immunocompromised patients – particularly in the HIV-infected – who present with subacute or chronic joint pain refractory to conventional treatment, osteoarticular tuberculosis should still be included in the differential diagnosis. We report on a lethal case of disseminated tuberculosis in an HIV-infected subject. Dissemination may have resulted from the implantation of an articular prosthesis in a knee joint with unsuspected osteoarticular tuberculosis. The diagnosis was established months later when the patient presented with far-advanced tuberculous meningitis, miliary tuberculosis of the lungs, femoral osteomyelitis and extended cold abscesses along the femoral shaft. Failure to respond to a conventional four-drug regimen is explained by the resistance pattern of his multi-drug resistant strain of Mycobacterium tuberculosis, which was only reported after the patients death. This case illustrates the diagnostic challenges of osteoarticular tuberculosis and the consequences of a diagnostic delay in an HIV-infected individual.

Determination of voriconazole in human serum and plasma by micellar electrokinetic chromatography

The micellar electrokinetic capillary chromatography (MEKC) separation and analysis of voriconazole and UK 115794 (internal standard) were examined and an assay for determination of voriconazole in human plasma and serum was developed. The MEKC medium comprises a 2:15 (v/v) mixture of methanol and a pH 9.3 buffer composed of 5mM Na(2)B(4)O(7), 7 mM Na(2)HPO(4) and 54 mM SDS. Sample preparation is based upon liquid/liquid extraction with ethylacetate and dichloromethane (75%/25%) at physiological pH. Using this approach with 250 microl serum or plasma and reconstitution of the dried extract into 100 microl of a buffer composed of 0.5mM Na(2)B(4)O(7) and 0.7 mM Na(2)HPO(4) (pH 9.3), the detection and quantitation limits were determined to be 0.1 and 0.2 microg/ml, respectively, a sensitivity that is suitable for therapeutic drug monitoring of voriconazole (provisional therapeutic range: 1-6 microg/ml) in human plasma and serum samples. The method was validated and compared to an HPLC method, showing excellent agreement between the two for a set of 91 samples that stemmed from patients being treated with voriconazole. The MEKC assay is also demonstrated to be suitable to explore pharmacokinetic data of voriconazole.