Brita Bruun

Semi-national surveillance of fungaemia in Denmark 2004–2006: increasing incidence of fungaemia and numbers of isolates with reduced azole susceptibility

A semi-national laboratory-based surveillance programme for fungaemia was initiated in 2003 that now covers c. 3.5 million inhabitants (64%) of the Danish population. In total, 1089 episodes of fungaemia were recorded during 2004-2006, corresponding to an annual incidence of 10.4/100 000 inhabitants. The annual number of episodes increased by 17% during the study period. Candida spp. accounted for 98% of the fungal pathogens. Although Candida albicans remained predominant, the proportion of C. albicans decreased from 66.1% in 2004 to 53.8% in 2006 (p <0.01), and varied considerably among participating departments, e.g., from 51.1% at a university hospital in Copenhagen to 67.6% in North Jutland County. Candida glabrata ranked second, and increased in proportion from 16.7% to 22.7% (p 0.04). Candida krusei was isolated rarely (4.1%), but the proportion doubled during the study period from 3.2% to 6.4% (p 0.06). MIC distributions of amphotericin B and caspofungin were in close agreement with the patterns predicted by species identification; however, decreased susceptibility to voriconazole, defined as an MIC of >1 mg/L, was detected in one (2.5%) C. glabrata isolate in 2004 and in 12 (14.0%) isolates in 2006 (p 0.03). Overall, the proportion of isolates with decreased susceptibility to fluconazole exceeded 30% in 2006. The incidence of fungaemia in Denmark was three-fold higher than that reported from other Nordic countries and is increasing. Decreased susceptibility to fluconazole is frequent, and a new trend towards C. glabrata isolates with elevated voriconazole MICs was observed.

Breakthrough Aspergillus fumigatus and Candida albicans Double Infection during Caspofungin Treatment: Laboratory Characteristics and Implication for Susceptibility Testing

ABSTRACT Caspofungin is used for the treatment of acute invasive candidiasis and as salvage treatment for invasive aspergillosis. We report characteristics of isolates of Candida albicans and Aspergillus fumigatus detected in a patient with breakthrough infection complicating severe gastrointestinal surgery and evaluate the capability of susceptibility methods to identify candin resistance. The susceptibility of C. albicans to caspofungin and anidulafungin was investigated by Etest, microdilution (European Committee on Antibiotic Susceptibility Testing [EUCAST] and CLSI), disk diffusion, agar dilution, and FKS1 sequencing and in a mouse model. Tissue was examined by immunohistochemistry, PCR, and sequencing for the presence of A. fumigatus and resistance mutations. The MICs for the C. albicans isolate were as follows: >32 μg/ml caspofungin and 0.5 μg/ml anidulafungin by Etest, 2 μg/ml caspofungin and 0.125 μg/ml anidulafungin by EUCAST methods, and 1 μg/ml caspofungin and 0.5 μg/ml anidulafungin by CLSI methods. Sequencing of the FKS1 gene revealed a mutation leading to an S645P substitution. Caspofungin and anidulafungin failed to reduce kidney CFU counts in animals inoculated with this isolate (P > 0.05 compared to untreated control animals), while both candins completely sterilized the kidneys in animals infected with a control isolate. Disk diffusion and agar dilution methods clearly separated the two isolates. Immunohistochemistry and sequencing confirmed the presence of A. fumigatus without FSK1 resistance mutations in liver and lung tissues. Breakthrough disseminated aspergillosis and candidiasis developed despite an absence of characteristic FKS1 resistance mutations in the Aspergillus isolates. EUCAST and CLSI methodology did not separate the candin-resistant clinical isolate from the sensitive control isolate as well as did the Etest and agar methods.

Xanthomonas maltophilia Bacteremia in Immunocompromised Hematological Patients

Epidemiological, microbiological and clinical characteristics of 14 episodes of Xanthomonas maltophilia bacteremia in 12 seriously immunocompromised hematological patients, admitted to Rigshospitalet in Copenhagen over the 3-year period 1989-91, were evaluated. The results were compared with a randomly selected control group of 25 patients with Escherichia coli bacteremia. Hospital acquired bacteremia was more common among the patients with X. maltophilia bacteremia (p < 0.01). Treatment with broad-spectrum antibiotics before the bacteremic episode was markedly more common among the patients with X. maltophilia bacteremia (p < 0.001). The presence of a central venous catheter and previous treatment with corticosteroids were more frequent in patients with X. maltophilia bacteremia (p < 0.05). The X. maltophilia blood culture isolates were generally resistant to aminoglycosides and most beta-lactams. The mortality rates related to bacteremia caused by X. maltophilia and E. coli were 14% and 20%, respectively.

Molecular screening for Candida orthopsilosis and Candida metapsilosis among Danish Candida parapsilosis group blood culture isolates: proposal of a new RFLP profile for differentiation.

Candida orthopsilosis and Candida metapsilosis are recently described species phenotypically indistinguishable from Candida parapsilosis . We evaluated phenotyping and molecular methods for the detection of these species among 79 unique blood culture isolates of the C. parapsilosis group obtained during the years 2004-2008. The isolates were screened by PCR amplification of the secondary alcohol dehydrogenase-encoding gene ( SADH) followed by digestion with the restriction enzyme Ban I, using C. parapsilosis ATCC 22019, C. orthopsilosis ATCC 96139 and C. metapsilosis ATCC 96144 as controls. Isolates with RFLP patterns distinct from C. parapsilosis were characterized by sequence analysis of the ITS1-ITS2, 26S rRNA (D1/D2) and SADH regions. Restriction patterns for the 3 species with each of 610 restriction enzymes were predicted in silico using 12 available sequences. By PCR-RFLP of the SADH gene alone, four isolates (5.1 %) had a pattern identical to the C. orthopsilosis reference strain. Sequence analysis of SADH and ITS (internal transcribed spacer) regions identified two of these isolates as C. metapsilosis. These results were confirmed by creating a phylogenetic tree based on concatenated sequences of SADH, ITS and 26S rRNA gene sequence regions. Optimal differentiation between C. parapsilosis, C. metapsilosis and C. orthopsilosis was predicted using digestion with NlaIII, producing discriminatory band sizes of: 131 and 505 bp; 74, 288 and 348 bp; and 131, 217 and 288 bp, respectively. This was confirmed using the reference strains and 79 clinical isolates. In conclusion, reliable discrimination was obtained by PCR-RFLP profile analysis of the SADH gene after digestion with NlaIII but not with BanI. C. metapsilosis and C. orthopsilosis are involved in a small but significant number of invasive infections in Denmark.

Non-typhoid Salmonella bacteraemia in greater Copenhagen 1984 to 1988

A retrospective survey of non-typhoidSalmonella bacteraemia in the period 1984 to 1988 was carried out by the five departments of clinical microbiology in Greater Copenhagen. A total of 168 patients were identified. A gradual increase was observed from 11 cases in 1984 to 58 cases in 1988. The corresponding incidence per 100,000 inhabitants in Copenhagen rose from 0.9 in 1984 to 5.0 in 1988. During the same period the total registered incidence of humanSalmonella infections in Denmark increased from 17.6 to 67.4 per 100,000 inhabitants. The serotype most often isolated from bacteraemic patients wasSalmonella dublin followed bySalmonella enteritidis andSalmonella typhimurium. Salmonella dublin demonstrated enhanced invasive and pathogenic properties. Predisposing factors were present in 56 % of the patients; the most common was malignant disease. A fatal or complicated course of the bacteraemia was observed more frequently in patients with underlying diseases than in persons who had previously been healthy. A total of 17 % of the patients died; one-fifth of these had a ruptured aortic aneurysm. It is concluded that the substantial increase in the number of cases and the often serious course taken by the infection demonstrate a need for increased efforts at prophylaxis.

Fungemia with Saccharomycetaceae: Report of four Cases and Review of the Literature

Invasive fungal disease with Saccharomycetaceae is very rarely reported and these fungi are usually considered nonpathogenic. We report here 4 cases of positive blood cultures with fungal species belonging to this family. Severe neutropenia, permanent central venous catheter, ongoing antibacterial chemotherapy, and major abdominal surgery were identified as risk factors for fungemia in patients. In the immunocompromised host isolation of such species from sterile fluids cannot be ignored but should be considered an opportunistic infection and treated as such.

Diversity of nosocomial Xanthomonas maltophilia (Stenotrophomonas maltophilia) as determined by ribotyping.

Seventy-seven clinical isolates ofXanthomonas maltophilia (Stenotrophomonas maltophilia) were consecutively collected from the Rigshospitalet, Copenhagen, ribotyped and compared with the ribotypes of 25 other clinical and reference strains ofXanthomonas maltophilia. Using restriction enzymeEcoRI, 20 different ribotypes were observed, with 78 isolates displaying the five most common types. Using another enzyme,BamHI, these 78 isolates were further subdivided into 16 different ribotypes. Three patients harboured two strains with different ribotypes. No type was found related to only one department and no single-strain outbreak was detected. The origin of this wealth of different strains in hospital patients needs to be established.

Flavobacterium meningosepticum infection in a neonatal ward

An outbreak of infections due toFlavobacterium meningosepticum type C in a neonatal intensive care unit is described. During a period of two weeks, two infants developed meningitis and a third was colonized in the respiratory tract and had transient bacteremia. The two meningitis patients were treated with clindamycin, rifampicin and cefotaxime systemically, plus rifampicin intraventricularly. Bacteriological eradication was achieved within 48 h, and both infants recovered from the meningitis without apparent neurological sequelae; however, one infant died two months later of unrelated causes. Environmental surveillance cultures failed to demonstrate a reservoir for the epidemic strain, but otherFlavobacterium strains were recovered. Two clinically healthy infants were found to be colonized in the nasopharynx with strains that were extremely difficult to differentiate phenotypically from the epidemic strain. Extensive characterization of strains is necessary in order to differentiate between strains and subsequently to determine a certain source of infection.

Bedside diagnosis of imported malaria using the Binax Now malaria antigen detection test

Malaria may be misdiagnosed in non-endemic countries when the necessary experience for rapid expert microscopy is lacking. Rapid diagnostic tests may improve the diagnosis and may play a role as a bedside diagnostic tool. In a multicentre study we recruited patients suspected of malaria over a period of 14 months. The Binax Now Malaria rapid test was used at the bedside and in the clinical microbiology laboratory. The training of clinical staff was monitored and their experience with the use of the test was recorded. 542 patients were included, 80 of whom had malaria diagnosed by microscopy. The rapid test used at the bedside had a sensitivity of 88% for the detection of P. falciparum compared to 95% when the test was performed in the microbiology laboratory. The risk of technical problems and invalid tests was highest when the test was used at the bedside. The rapid diagnostic test may be useful for the diagnosis of P. falciparum malaria when used by routine laboratory staff, but could lead to misdiagnoses when used at the bedside. Microscopy is still essential in order to identify the few missed diagnoses, to determine the degree of parasitaemia, and to ensure species diagnosis, including mixed infections.

Changing etiology of bacteremia in patients with hematological malignancies in Denmark

To ascertain whether the microbiological etiology of bacteremia among patients with hematological malignancies has changed in Denmark, the species distribution of clinically relevant blood culture isolates from the Hematological Department at Rigshospitalet, Copenhagen in 1990 was compared with 2 previous studies (1970-72; 1981-85). In addition, time trends of the etiology of bacteremia among hematological patients in Copenhagen (eastern Denmark) and in Arhus (western Denmark) were compared. In contrast to many other studies, a significant increase in the proportion of Gram-negative aerobes was observed in Copenhagen (from 43% in 1981-85 to 55% in 1990; p < 0.05), whereas in Arhus the proportion of Gram-positive aerobes increased steadily during the 1980s (from 34% to 51%; p < 0.05). In Copenhagen, non-hemolytic streptococci and Xanthomonas maltophilia increased significantly and accounted for 10% (p < 0.01) and 5% (p < 0.05) respectively, of all isolates in 1990, whereas Staphylococcus aureus during the 2 decades studied decreased from 25% to 8% (p < 0.001). In both regions, a decrease was observed in the proportion of Pseudomonas aeruginosa which accounted for only about 5% of all isolates in 1990. No changes were observed in the rates of anaerobes and yeasts. Several factors may contribute to the reported differences in the etiology of bacteremia among hematological patients, e.g. criteria used to assign the clinical significance of the isolate, blood culture system used, practice of using indwelling intravenous catheters, different policies with respect to antimicrobial treatment, and the degree of immunosuppression. A local surveillance of blood culture isolates is mandatory if changes in etiology and resistance development are to be detected.