Close association between oropharyngeal and rhinopharyngeal colonization with Staphylococcus aureus - clues to new insight of MRSA colonization of the oropharynx.

This study provides data on prevalence of Staphylococcus aureus in oropharynx, rhinopharynx and vestibulum nasi. Specimens were taken from these three pharyngeal sites in 346 patients and analysed for S. aureus. Abnormal pharyngeal findings and patient histories were recorded. S. aureus was found in 8.1%, 7.2% and 20.2% of all specimens from oropharynx, rhinopharynx and vestibulum nasi, respectively. A strong association between colonization of oropharynx and rhinopharynx was found, especially when vestibulum nasi was not colonized. These findings can be used in development of more effective meticillin-resistant Staphylococcus aureus decolonization regimes.

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.

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.

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.

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.

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.

Problems in identification of Francisella philomiragia associated with fatal bacteremia in a patient with chronic granulomatous disease.

Francisella philomiragia is a rare gram-negative, halophilic coccobacillus with bizarre spherical forms on primary isolation. A case of F. philomiragia bacteremia in a 24-year-old patient with chronic granulomatous disease is reported. Identification of F. philomiragia was problematic with conventional tests but was done correctly and rapidly by kit 16S ribosomal DNA sequencing.

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

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.

Clinical significance and taxonomy of Actinobacillus hominis.

Clinical findings in 36 immunosuppressed patients with lower respiratory tract infection or bacteremia with Actinobacillus hominis are described. Animal contact was only recorded for three patients; nine patients died despite appropriate antimicrobial treatment. Although infections with this microorganism seem to be rare, the fact that 37 of 46 strains characterized in this study have been found in Copenhagen indicates that under-reporting may occur. A. hominis is phenotypically relatively homogeneous but can be difficult to differentiate from other Actinobacillus species unless extensive biochemical testing is performed. Mannose-positive strains of A. hominis are especially difficult to differentiate from A. equuli. Attempts to identify A. hominis by automatic identification systems may lead to misidentifications. Ribotyping and DNA-DNA hybridization data show that A. hominis is a homogeneous species clearly separated from other species within the genus Actinobacillus.

Effects of targeting herpes simplex virus type 1 gD to the endoplasmic reticulum and trans-Golgi network.

Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) was modified to encode targeting signals known to localize proteins to either the endoplasmic reticulum (ER) or the trans-Golgi network. These motifs conferred the predicted targeting properties on gD in transfected cells as judged by immunofluorescence staining, and the exclusion of targeted gD from the cell surface was confirmed by the fact that these molecules exhibited substantially reduced activity in cell-cell fusion assays. Recombinant viruses expressing Golgi-targeted forms of gD grew to wild-type levels in noncomplementing cells, exhibited unaltered particle/infectivity ratios, and were found to contain wild-type levels of gD, whereas a recombinant expressing ER-retained gD was helper cell dependent and, when grown on noncomplementing cells, produced virions of low specific infectivity with greatly reduced levels of gD. These data imply that HSV-1 acquires its final membrane from a post-ER compartment and lend support to the view that the virus undergoes de-envelopment and reenvelopment steps during virus egress.

Unexpected cross-reaction with Fusobacterium necrophorum in a PCR for detection of mycoplasmas.

Cross-reactions with Fusobacterium necrophorum were found in a PCR designed for detection of a wide range of mycoplasma species. Twenty-five strains of Fusobacterium were examined; all 14 F. necrophorum strains reacted positively, whereas all 7 Fusobacterium nucleatum strains reacted negatively. Two strains that were not F. necrophorum yielded variable results.

Ribotyping of strains of Moraxella (Branhamella) catarrhalis cultured from the nasopharynx and middle ear of children with otitis media.

Moraxella (Branhaomella) catarrhalis is frequently present in the nasopharyngeal microflora of small children, especially during episodes of acute otitis media . By means of ribotyping (restriction endonuclease analysis of chromosomal DNA combined with rRNA probing), we studied the genetic heterogeneity of 78 cultures of M. catarrhalis obtained from different localities in the nasopharynx of nine young children with secretory otitis media. Using HindIII and PstI as endonucleases, five different ribotypes were recognized, representing at least five different genotypes of M. catarrhalis. The distribution of these types was found to be almost identical to the distribution among 16 M. catarrhalis strains cultured from middle ear exudates of 16 children with acute otitis media. Ribotype HAPA was found in two-thirds of all the cultures investigated, and 44% of the children harboured more than one ribotype in the nasopharynx at the same time. The vast majority of the nasopharyngeal M. catarrhalis cultures were beta-lactamase positive. One child had both a HAPA ribotype, beta-lactamase-negative strain in the nasopharyngeal secretions, and HAPA ribotype, beta-lactamase-positive strains at the entrance of the eustachian tube, the nasopharyngeal tonsils, the folds of the nasopharyngeal tonsils and the oropharynx. All except one of the M. catarrhalis strains cultured from middle ear exudates were beta-lactamase positive.

Glycoproteins gB, gD, and gHgL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system.

Herpes simplex virus type 1 glycoproteins gB, gD, and gHgL were expressed by transient transfection of Cos cells. Polykaryocyte formation above the background level seen in untransfected controls was observed only if all three components were expressed. Thus, gB, gD, and gHgL are necessary and sufficient to induce membrane fusion.

Problems of identification in clinical microbiology exemplified by pig bite wound infections.

Our experience from attempts to identify bacteria isolated from boar bite/gore wounds is the background for a discussion of identification problems. Some organisms, although not very common or well-known, can be identified when using commercial kits or conventional methods, provided they are sufficiently characterized, as exemplified by Pasteurella aerogenes isolated from cases 1 and 2. Some organisms may be wrongly identified, or not identified, by both commercial kits and conventional methods, unless seen by experienced microbiologists with knowledge of the original literature. This is exemplified by case 3, in which the final identification result was Bisgaard's taxon 15. Sometimes isolates cannot be identified even in reference laboratories and by using available identification tables and databases. In such cases, the organism involved may turn out to belong to a previously undescribed taxon. This is illustrated by the strains isolated from cases 4 and 5.

Site-directed and linker insertion mutagenesis of herpes simplex virus type 1 glycoprotein H.

The gH-gL complex of herpes simplex virus type 1 (HSV-1) is essential for virion infectivity and virus-induced cell fusion, but functional domains of the gH molecule remain to be defined. We have addressed this question by mutagenesis. A set of linker insertion mutants in HSV-1 gH was generated and tested in transient assays for their ability to complement a gH-negative virus. Insertions at three sites in the C-terminal third of the external domain affected the ability of gH to function in cell-cell fusion and virus entry, while insertions at six sites in the N-terminal half of the external domain induced conformational changes in gH such that it was not recognized by monoclonal antibody LP11, although expression at the cell surface was unchanged. A recombinant virus in which a potential integrin-binding motif, RGD, in gH was changed to the triplet RGE entered cells as efficiently as the wild type, indicating that HSV-1 entry is not mediated by means of the gH-RGD motif binding to cell surface integrins. Furthermore, mutagenesis of the glycosylation site which is positionally conserved in all herpesvirus gH sequences in close proximity to the transmembrane domain generated a recombinant virus that grew in vitro with wild-type single-step kinetics.