Differentiation of Candida glabrata, C. nivariensis and C. bracarensis based on fragment length polymorphism of ITS1 and ITS2 and restriction fragment length polymorphism of ITS and D1/D2 regions in rDNA.

Different molecular methods for the discrimination of Candida glabrata, C. bracarensis and C. nivariensis were evaluated and the prevalence of these species among Danish blood isolates investigated. Control strains were used to determine fragment length polymorphism in the ITS1, ITS2, ITS1-5.8S-ITS2 regions and in the D1/D2 domain of 26S rDNA using primers designed for this study. A total of 133 blood isolates previously identified as C. glabrata were examined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the peptide nucleic acid-fluorescent in situ hybridization (PNA-FISH) method. The size of ITS1 allowed differentiation between C. glabrata (483), C. nivariensis (361) and C. bracarensis (385), whereas the ITS2 region was of similar size in C. nivariensis (417) and C. glabrata (418). Sequence analysis of the ITS region suggested that many restriction enzymes were suitable for RFLP differentiation of the species. Enzymatic digestion of the D1/D2 domain with TatI produced unique band sizes for each of the three species. PCR-RFLP and PNA-FISH were in agreement for all of the isolates tested. None of the 133 Danish blood isolates were C. nivariensis or C. bracarensis. Fragment size polymorphism of ITS1 and RFLP of the D1/D2 domain or the ITS region are useful methods for the differentiation of the species within the C. glabrata group. C. bracarensis and C. nivariensis are rare among Danish C. glabrata blood isolates.

Social science perspectives on schistosomiasis control in Africa: past trends and future directions.

New ways of integrating and scaling up control of neglected tropical diseases (including schistosomiasis) are presently underway. In this context consideration of social science perspectives is essential. In this article, we review social science publications of relevance to sustained control of schistosomiasis in Africa including diagnosis and screening, treatment, supply of clean water and improved sanitation, as well as health communication. Studies of community involvement and links between schistosomiasis control programmes and broader health care systems are also explored. Directions for future social science of relevance to sustainable schistosomiasis control are outlined, including ways of ensuring equitable access to health services as well as involvement of endemic communities and local health care systems based on equal partnership.

Outbreak of Clostridium difficile 027 in North Zealand, Denmark, 2008-2009.

We report an outbreak of Clostridium difficile PCR ribotype 027 in Denmark. The outbreak includes to date 73 cases from the area north of Copenhagen, but there may be related cases elsewhere in Zealand. Most infections are healthcare-associated and in patients who previously received antibiotic treatment. The strain is resistant to moxifloxacin, erythromycin, and clindamycin, and carries genes for toxin A, toxin B, and for the binary toxin. The antimicrobial pattern differs from that of the strain involved in a small cluster in Denmark in 2006-2007. Because of this outbreak, hygienic measures in the involved hospitals have been reinforced. Nationwide, microbiological laboratories were alerted to the outbreak and encouraged to send isolates for toxin profiling and PCR ribotyping.

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.

Shewanella algae and Shewanella putrefaciens: clinical and microbiological characteristics.

The occurrence of the two Shewanella species found in clinical specimens, Shewanella algae and Shewanella putrefaciens, correlates with the temperature and salinity of seawater. This means that Shewanella infections occur in warm climates or during especially warm summers in temperate climates. The infections described most commonly involve ears, skin and soft tissue, with or without bacteraemia. Primary bacteraemia with a fulminant course is also seen in immunocompromised patients. Important differential characteristics between the two species include the ability of S. algae to produce mucoid colonies with beta-haemolysis on sheep blood agar, to grow at 42 degrees C and in NaCl 6% w/v, and to reduce nitrite, and an inability to produce acid from maltose, all of which are in contrast to the characteristics of S. putrefaciens. Automated identification systems fail to differentiate between S. algae and S. putrefaciens, as S. algae is not included in the databases of these systems. Presumably for this reason, most Shewanella infections reported during recent years have been attributed to S. putrefaciens. However, when extensive phenotypic characterisation is performed, most human infections are seen to be caused by S. algae. As the two species seem to have different pathogenic potential for humans, correct identification is important, and this is possible in routine clinical microbiology laboratories.

Invasive listeriosis in Denmark 1994-2003: a review of 299 cases with special emphasis on risk factors for mortality.

Listeriosis is a rare, but serious, foodborne infection which, in the invasive form, presents as bloodstream (BS) infection, an infection of the central nervous system (CNS), a maternofetal infection or a focal infection. The disease is notifiable in Denmark. This paper reviews the results of the Danish surveillance of invasive listeriosis from 1994 to 2003, excluding maternofetal cases. In total, 299 invasive cases of listeriosis were reported. Two-thirds of the cases were caused by isolates of serogroup 1/2, and one-third by serogroup 4. Most (70%) cases had conditions known to predispose to listeriosis. More patients with BS infection were predisposed because of concurrent underlying illness than were patients with CNS infection. Half of the patients were aged > 70 years, and 21% died of the disease. There was no change in the case fatality rate (CFR) during the 10-year period. The CFR was identical for men and women. BS and CNS infection caused the same incidence of mortality, but no mortality was observed in patients with focal infections at normally sterile body sites. In a multivariate analysis, isolates belonging to serogroup 4 were associated with a higher CFR than were isolates of serogroup 1/2. In patients aged < 70 years, underlying conditions predisposing to disease were related strongly to mortality, which was not the case in patients aged > 70 years. The underlying conditions associated most strongly with mortality in the younger age group were non-haematological malignancies.

Infections in patients treated with high-dose chemotherapy for germ cell tumours.

25 patients with disseminated germ cell tumours were treated with high-dose cisplatin and etoposide (40 mg/m2 and 200 mg/m2 daily x five, respectively) leading to severe myelosuppression. A comprehensive study was undertaken in order to identify and describe the bacterial, viral and fungal infections in this group of patients. Fever (> 38.5 degrees C) and leucopenia (white blood cell count < 1.0 x 10(9)/l) were observed in 61 of 90 treatment cycles (68%). A microbiological aetiology compatible with the clinical manifestations of infection could be identified in 33 of the 61 febrile episodes (54%). Bacteraemia occurred in 14 episodes in 12 patients. Eight episodes (57%) involved gram-positive aerobic bacteria.

Ceftriaxone versus latamoxef in febrile neutropenic patients: empirical monotherapy in patients with solid tumours.

121 patients with 132 febrile episodes were randomised to ceftriaxone or latamoxef monotherapy in order to compare antibiotic efficacy in neutropenic patients treated with cytotoxic chemotherapy for solid tumours. In 80 evaluable episodes no significant differences were observed between the two groups with respect to efficacy and fatal failure rates. Of episodes treated with ceftriaxone, 67% showed a favourable clinical response vs. 61% in the latamoxef group. The clinical response rates in episodes with documented bacterial infections were 67 and 56% in the two treatment groups. In 18% of the episodes with documented initial infections the patients died of presumably uncontrolled infection. The convenient once daily dosage schedule combined with fewer severe adverse reactions favours the use of ceftriaxone instead of latamoxef. Although a relative high degree of response was seen, empirical antibiotic monotherapy apparently does not offer a sufficient antibacterial cover in infections in this type of patient with defective host immunity.

Genotypic heterogeneity of Flavobacterium group IIb and Flavobacterium breve demonstrated by DNA-DNA hybridization.

DNA-DNA hybridization studies on 42 stains presumptively identified as members of Flavobacterium group IIb and Flavobacterium breve indicated pronounced genotypic heterogeneity within these taxa. Three large groups highly related to the type strains of F. gleum, F. indologenes and F. breve respectively, and eight small groups were found. The group containing the type strain of F. breve was phenotypically indistinguishable from another genomic group, and these two groups were significantly separated from the other flavobacteria studied. The other nine genomic groups, representing Flavobacterium group IIb, could not with certainty be differentiated from each other by phenotypic characteristics, and there is no evidence indicating that these genomic groups differ from each other with respect to pathogenicity or ecology. Thus, it is suggested that for the time being the name "Flavobacterium group IIb" rather than specific epithets continue to be used for these bacteria.

Crossed immunoelectrophoretic analysis of Flavobacterium meningosepticum DNA hybridization groups I and II.

By means of crossed immunoelectrophoresis and crossed-line immunoelectrophoresis, antigens were compared from four strains and five strains, respectively, of Flavobacterium meningosepticum DNA hybridization groups I and II. Two reference antisera raised against the pooled antigens of each of the two DNA groups were used. The two reference antigen-antibody systems produced 81 and 51 immunoprecipitates, respectively, and within each group nearly all antigens were common to all strains, giving matching coefficients of 0.98-1.00. Group specific antigens were not detected. When antigens from individual strains from each group were compared to individual strains of the other group, however, 5-15 of the antigens were not cross-reactive, giving matching coefficients of 0.83-0.94. The antigenic relatedness between strains belonging to the two different groups was, therefore, just below the level commonly found for strains belonging to the same species. The results thus confirm the previously published differences detected by a DNA hybridization technique.

Neisseria meningitidis: occurrence in non-pneumonic pulmonary infections.

A two-year survey at our hospital revealed 19 patients suspected of pulmonary infection from whom Neisseria meningitidis was isolated from lower respiratory tract specimens, in 17 cases in pure culture. Of the isolates, 16, two and one belonged to serogroup B, C and 29E, respectively. All except three patients suffered from chronic pulmonary disease, notably chronic bronchitis. It was found likely or possible that N. meningitidis was the causative agent in 12 patients with symptoms of acute bronchitis. Results of tests for complement-fixing antibodies against N. meningitidis (MAT) were generally not found to be useful when assessing the possible causative role of N. meningitidis in non-pneumonic pulmonary infections.

Antimicrobial susceptibility and beta-lactamase characterization of Branhamella catarrhalis isolates from 1983/1984 and 1988.

Branhamella catarrhalis isolates from lower respiratory tract specimens collected in 1983/84 (n = 50) and 1988 (n = 30) were examined for beta-lactamase production. The percent of beta-lactamase-producing strains increased from 25% to 63% from 1983/84 to 1988. beta-lactamases from 30 strains could be typed, and of these 28 were of the BRO-1 type and two the BRO-2 type. The two beta-lactamase inhibitors, clavulanic acid and brobactam were very active against beta-lactamase extracts whether of the BRO-1 or the BRO-2 type. Susceptibility to a number of antimicrobial agents or combinations of agents was determined by a plate dilution method (MICs) and by a tablet diffusion method. The penicillin tablet (Rosco Neo-Sensitabs) was found to be useful in discriminating between beta-lactamase-producing and non-beta-lactamase- producing strains of B. catarrhalis.

In vitro susceptibility of 124 Xanthomonas maltophilia (Stenotrophomonas maltophilia) isolates: comparison of the agar dilution method with the E-test and two agar diffusion methods.

The in vitro susceptibility of 124 Xanthomonas maltophilia isolates was tested by four methods: Agar dilution (reference method), E-test, a disk diffusion and a tablet diffusion method. Trimethoprim-sulfamethoxazole had the highest activity against X. maltophilia, followed by a combination of aztreonam-clavulanic acid at different ratios, the ratio 1:1 being the most active with a susceptibility rate of 85% as compared to 2% for aztreonam alone. Addition of the beta-lactamase inhibitor tazobactam to piperacillin enhanced the rate of susceptible isolates from 31% to 53%, Relatively few isolates were susceptible to ciprofloxacin (27%) and gentamicin (9%). Generally, the disk diffusion method had a considerably higher frequency of "very major" discrepancies when compared with the agar dilution method than with the other methods. The susceptibility of X. maltophilia to trimethoprim-sulfamethoxazole and ciprofloxacin could reliably be determined by all the diffusion methods tested, but otherwise the agar dilution method is to be preferred. A standardized and reliable diffusion method for susceptibility testing of X. maltophilia remains to be found. Trimethoprim-sulfamethoxazole must be considered the drug of choice in the treatment of severe X. maltophilia infections. The combination aztreonam-clavulanic acid is promising, but must be proved in a clinical setting.

Genotypic and phenotypic relatedness of 80 strains of Branhamella catarrhalis of worldwide origin.

80 clinical Branhamella catarrhalis strains of worldwide origin were examined for genotypic relatedness and phenotypic characteristics. Using a quantitative bacterial dot method for DNA-DNA hybridization the strains were found to form a homogeneous group with delta Tm-values ranging from 0.0-2.3 degrees C, In Minibact-N, an identification kit for oxidase positive, Gram-negative diplococci using eight phenotypic characteristics, all isolates were correctly identified and also demonstrated complete homogeneity except for beta-lactamase production. Type strains representing the genera Branhamella, Moraxella and Neisseria were also examined for comparison. B. catarrhalis strain NCTC 4103-known to be atypical-had a delta Tm-value of 5.7 degrees C and produced gamma-glutamylaminopeptidase, in contrast to all other B. catarrhalis strains. In GN MicroPlate, a kit which tests utilizable carbon sources, B. catarrhalis strains were found to be able to utilize up to 16 to 95 carbon sources.

Mecillinam susceptibility as an indicator of beta-lactamase production in Staphylococcus aureus.

The use of direct susceptibility testing from specimens has led to the fortuitous observation that penicillin-susceptible strains have larger inhibition zones for mecillinam than do beta-lactamase producers. The current study was, therefore, undertaken to test 179 Staphylococcus aureus isolates for mecillinam susceptibility by Rosco Neo-Sensitabs and to compare the results with commonly used tests for beta-lactamase production, i.e. size and character of penicillin inhibition zones, chromogenic cephalosporin (nitrocefin) tests and clover leaf assays. Agreement between methods was reached for 175 of 179 strains when disregarding the results of the nitrocefin tests, 88 isolates being found susceptible and 87 being found to be beta-lactamase producers. All 88 susceptible isolates had mecillinam zones of >22 mm, with the great majority being >25 mm; double zones did, however, occur. The 87 beta-lactamase producers had zones <14 mm or no zones. Four isolates presenting problems in had mecillinam zones of

Fusarium fungaemia in immunocompromised patients.

Fusarium spp. cause infections only rarely in immunologically competent hosts, but disseminated infection may occur in severely immunocompromised patients. Symptoms of disseminated infection are persistent fever, despite broad-spectrum antibacterial and antifungal treatment, associated with skin lesions, most commonly on the extremities, in 60-80% of patients. A mortality rate of 50-75% has been reported for patients with disseminated fusariosis. Despite treatment failures, amphotericin B remains the preferred drug, in part because of lack of alternatives. Voriconazole is a promising new agent, but more clinical experience is required.

Bactec 9240 blood culture system: to preincubate at 35 degrees C or not?

Bactec Plus blood culture bottles were preincubated at 35 degrees C or at room temperature before entry into the Bactec 9240 instrument to determine the influence of preincubation temperature and time. Of 463 positive blood culture sets, 956 bottles were positive, of which the instrument detected 92.1%. Of 76 positive bottles undetected by the instrument, 68 were preincubated at 35 degrees C and eight at room temperature. The median entry delay and instrument detection times were 17.9 and 7.2 h for preincubated bottles, and 16.4 and 13.4 h for bottles held at room temperature. Short entry delay and inspection before entry into the instrument are necessary if preincubation at 35 degrees C is used.

Direct identification and susceptibility testing of enteric bacilli from positive blood cultures using VITEK (GNI+/GNS-GA).

OBJECTIVE: To study the possibility of reporting results of identification and susceptibility testing of Gram-negative bacilli the same day as bacteremia is detected by using direct inoculation from positive blood cultures (Bactec 9240) into VITEK GNI+ and GNS-GA cards. METHODS: All blood cultures with Gram-negative enteric bacillus-like morphology on microscopy found to be positive on workdays between 15 June 1999 and 29 February 2000 were included. Identification and susceptibility testing were done by three methods: the direct method using a suspension made by differential centrifugation of positive blood culture broth for inoculation of the VITEK cards; the standard method using an inoculum made from an overnight culture on a solid media; and the routine method (reference method) using conventional testing. RESULTS: Of 169 isolates, the direct method resulted in 75% correct identifications, 9% misidentifications and 17% non-identifications. All misidentified isolates were Escherichia coli, of which 80% were reported as Salmonella arizonae. Five biochemical tests yielded most of the aberrant results; correcting the citrate and malonate reactions in most cases led to correct identification by the VITEK database. Despite a negative H2S reaction, 11 E. coli isolates were reported as S. arizonae. Two-thirds (69%) of identifications were reported within 6 h, and 95% of these were correct. The direct susceptibility testing method was assessable for 140 isolates. Correct results were found in 99% of isolate-antimicrobial combinations, and 85% were reported within 6 h. CONCLUSION: The direct VITEK method could correctly report identifications and susceptibility patterns within 6 h, making same-day reporting possible for almost two-thirds (63%) of bacteremic episodes with Gram-negative bacilli. These results could probably be improved by modification of the identification algorithms of the VITEK software.

Moraxella (Branhamella) catarrhalis: restriction enzyme analysis typing with HinfI, HaeIII and PstI.

Restriction enzyme analysis typing with HinfI, HaeIII and PstI was performed on Moraxella (Branhamella) catarrhalis strains consecutively collected from children suspected of respiratory tract infection and the type strain. Use of HinfI gave the most distinct patterns. Great polymorphism was seen among strains.

Catheter-related sepsis in long-term parenteral nutrition with Broviac catheters. An evaluation of different disinfectants.

From April 1976 to January 1988, 58 patients received home parenteral nutrition for 2-138 months, median 36 months, corresponding to a total treatment period of 233 patient years. Before 1980 and after 1985, 0.5-2% iodine tincture or 0.5% chlorhexidine in 70% ethyl alcohol were used to disinfect the exit site of the catheter and the connections of the infusion line. In these periods the sepsis incidence was 0.25-0.28 per catheter year, corresponding to one episode of sepsis per 3.6-4.0 catheter years. In the period 1980 to 1985, 10% povidone-iodine (Isobetadine) was used, and the incidence in this period was 0.58, corresponding to one episode of sepsis per 1.7 catheter year. This suggests that 10% povidone-iodine may be inferior to iodine-tincture and chlorhexidine alcohol in this type of catheter care. The incidence of catheter sepsis was 0.32 per catheter year when the catheter was placed on the chest and 0.86 per catheter year with the catheter on the thigh. Klebsiella pneumoniae was the most common microorganism grown when the catheter was placed on the thigh, while coagulase-negative staphylococci were most common when the catheter was placed on the chest.