ALBERT

Notes: [Auszug] To the editor Campylobacter jejuni is the most frequent cause of bacterial diarrhea, which leads to the Guillain–Barré syndrome (GBS) or the Miller Fisher syndrome (MFS) in 1 in every 1000 infections. Neuropathy is probably triggered by molecular mimicry between C. jejuni lipooligo- ...

Notes: A group of patients (n=86) suffering from superficial abscesses was recruited in the Khartoum Teaching Hospital, Sudan. Detailed clinical and socio-economic data were collected. It appeared that 83% of all patients were younger than 40. Labourers were most prevalent (28%), followed by students (23%) and housewives (16%). The head and neck were most often affected (22%), with hands being second (19%). In 92% of all pus cultures a microbial agents was identified, the large majority being Staphylococcus aureus (69%). Among patients, 47% were nasal carriers of S. aureus, similar to the carriage rate measured among controls, suggesting that nasal carriage is no risk factor for abscess development. Multivariate logistic regression analysis revealed that a history of abscess, recent traditional medical treatment, poor hygiene and low socio-economic status were significantly and independently associated with the occurrence of superficial abscesses.

Notes: The nature of the micro-flora present in sputa of six different cystic fibrosis (CF) patients was assessed using routine microbiological culture and molecular methods. Bacterial genes for the small subunit ribosomal RNA (ssu rDNA) were specifically amplified from DNA extracted from the sputum samples, cloned and characterised by hybridisation and DNA sequencing. A large number of clones from six sputa were screened. Initially, oligonucleotide hybridisation was performed with five probes, specific for Gram-positives and Gram-negatives in general and the main pathogens for the CF patient (Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae). For a single sputum sample, the results were fully congruent when culture and molecular methods were compared. In the other five sputa, discrepancies for S. aureus and/or H. influenzae were documented. Although S. aureus DNA and H. influenzae DNA was detected in three and four sputa, respectively, strains could not be cultured. Although the PCR approach is not capable of distinguishing viable from dead bacteria, all of the CF patients had a history of S. aureus infections, while one of the CF patients once had cultivable H. influenzae in the sputum as well. A number of clones for probe-unidentified Gram-negative or Gram-positive bacterial species were further analysed by sequencing and additional potential pathogens were identified. Although routine culture of sputum frequently points to mono-specific exacerbations, our molecular data indicate that the other CF-related pathogens appear to be persistently present as well. We conclude that routine culture for bacterial pathogens from CF sputa yields limited microbiological information since it frequently fails to identify a number of pathogenic bacterial species that are potentially present in a viable status in the lungs of these patients.

Notes: Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.

Notes: Serial sputum isolates of Haemophilus influenzae (n= 69) were obtained from eight patients suffering from cystic fibrosis. For two of these patients all strains were analysed for polymorphism in the major outer membrane protein profile. For all patients the strains were genetically characterised by random amplification of polymorphic DNA analysis. All strains were included in a survey for polymorphism in regions containing moieties of repetitive DNA as well. A single locus containing trinucleotide repeat units, three loci harbouring tetranucleotides, one region comprising pentanucleotide units and two hexanucleotide repeat unit-containing loci were analysed for repeat number variability. Most of the regions were previously shown to be directly adjacent to or even within virulence genes. All regions behaved as genuine variable number of tandem repeat loci in the sense that genetic polymorphism based on the presence of varying numbers of repeat units could be demonstrated among different strains. Interestingly, several of the repeats showed variation in the absence of the variability as assessed by major outer membrane protein or random amplification of polymorphic DNA analysis. These observations indicate that the repeat loci may vary independently from major chromosomal polymorphism. Consequently, H. influenzae appears to modify its virulence gene regions of the chromosome during persistent colonisation of the lung in cystic fibrosis patients.

Notes: Moraxella catarrhalis is a bacterial species that has been implicated in 15–20% of all cases of otitis media in the USA and the complement-resistant variant of M. catarrhalis has been considered particularly pathogenic. A collection of geographically diverse, complement-sensitive (n=28) and -resistant strains (n=47) of M. catarrhalis was assembled in order to analyse the bacterial population structure. All strains were identified as M. catarrhalis by conventional microbiological and biochemical methods. Amplification of the small subunit (ssu) ribosomal RNA gene followed by restriction fragment length polymorphism (RFLP) analysis did not reveal consistent differences between serum-susceptible and -resistant M. catarrhalis isolates. Interestingly, upon automated ribotyping using the Qualicon RiboPrinter® microbial characterisation system, the complement-sensitive and -resistant strains segregated into two groups. This suggested the existence of two clearly distinguishable lineages within the species M. catarrhalis. This observation was corroborated by pulsed field gel electrophoresis (PFGE) of DNA macro-restriction fragments, a non-ribosomal PCR RFLP procedure and random amplification of polymorphic DNA (RAPD) analysis. All procedures grouped the two variants similarly. Redefinition of the taxonomic status of complement-resistant M. catarrhalis or even the definition of a new species may be opportune.

Notes: Swabs from the posterior vaginal fornix were obtained from 804 consecutive female patients visiting a large Dutch sexually transmitted diseases (STD) outpatient clinic. A detailed clinical history was obtained and complaints concerning the lower genital tract, such as vaginal discharge or vulval and vaginal irritation, were recorded. Patients were examined and the presence of non-physiological vaginal secretions was established by speculum examination. The swabs were monitored for bacterial vaginosis (BV) or Candida albicans infection. PCR diagnosis of Chlamydia trachomatis and Trichomonas vaginalis was performed as well. Four groups of patients (n=14–21) with BV or single infections caused by one of these three pathogens and a control group with no pathogens were selected and Mycoplasma hominis PCR was performed additionally. At clinical presentation, controls and single-infected patient groups were comparable with regard to complaints of the lower genital tract and sexual risk behavior defined as having prior STDs and/or admitted prostitution. Only in the T. vaginalis-positive group significantly more women reporting sexual risk behavior were found than in controls. In agreement with former in vitro observations, an in vivo association between the PCR-detected presence of M. hominis and T. vaginalis was established. In 79% of all samples positive for T. vaginalis, M. hominis could be detected, as compared to only 6% in control samples (P=0.0004). However, since single infections by either of the two pathogens were regularly observed, there does not seem to be an exclusive association between the species, as the bacterium is also more frequently found in cases of BV (P=0.026). Co-infection of M. hominis with C. albicans (11%) or C. trachomatis (0%) did not differ significantly from controls (6%). M. hominis did not associate with complaints of the lower genital tract. However, if all groups were combined there appears to be a very significant association between the presence of M. hominis and sexual risk behavior (P=0.0004). M. hominis and sexual risk behavior were more closely associated than M. hominis and T. vaginalis. No indications were found for an enhanced pathogenicity by either of the symbionts.

Notes: Coagulase negative staphylococci (CoNS) are a main cause of catheter related infections (CRI). Earlier studies (1994–1996) revealed a high incidence of CRI (6 per 1000 catheter days) among neutropenic hemato-oncologic patients in the Erasmus MC Hematology Department (Rotterdam, The Netherlands). This was mainly explained by expansion of two methicillin resistant Staphylococcus epidermidis (MRSE) clones (Nouwen et al., J. Clin. Microbiol. 36 (1998) 2696–2702). In a new, 16-bed unit in the same institution, we investigated the effect of strict clinical isolation measures on the incidence of CRI. During two 6-month screening periods (period I: April 1998–December 1998 and period II: April 1999–October 1999) all patients receiving a central venous catheter were prospectively monitored for the development of CRI. During period I every visitor of the cubicles had to wear hair caps, masks, gowns and gloves. During period II these procedures were abolished, but hands were cleansed using alcohol and masks were worn during both periods in case of coughing and sneezing. All CoNS strains isolated from blood cultures were genetically classifies by pulsed field gel electrophoresis (PFGE). The incidence of CRI during period I was 13.0 per 1000 catheter days, in comparison to 9.6 in period II (P=0.84). During this latter period, 19 CRI were diagnosed, 14 catheter related bacteremia episodes (CRB) and five local infections. Seventy-two percent (n=9) of CRB were due to a CoNS. The mean catheter survival until appearance of a CRI increased from 43 days during period I to 78 days in period II (P=0.39). The mean catheter survival until infection related removal was increased from 43 days to 133 days (P=0.12). During period I less experienced intervention radiologists introduced the catheters, which may have limited the efficacy of the strict hygiene measures. Thus, abolishing strict isolation precautions had no negative effect on the incidence of CRI. After genotyping of 38 MRSE strains isolated from blood and central venous catheter cultures of 12 patients in period II, eight PFGE types were found. Three types were found in more than one patient, but based on epidemiological data patient-to-patient spread could not be proven. No genotypic identity between patient and personnel CoNS isolates was shown and the two major clonal types that were present between 1994 and 1996 were not encountered. However, from December 1998 onwards new MRSE clones could be identified (types E and J). In conclusion, despite a constant rate of CRI and implementation of optimal patient care, clonal spread of MRSE strains was not prevented by strict hygiene measures.

Notes: The vitamin D endocrine system has been shown to influence the immune response and polymorphisms in the vitamin D receptor (VDR) gene have been associated with susceptibility to infectious diseases. We determined if the Cdx2, Fok I and Bsm I–Apa I–Taq I polymorphisms in the VDR gene were associated with nasal carriage of Staphylococcal aureus. We defined the S. aureus nasal carriage status (persistent, intermittent or non-carriage) for a group of more that 2000 elderly volunteers. The prevalence of persistent S. aureus nasal carriage was 18%, which was, however, not associated with any of the variant VDR genotypes. Our study into genetic determinants of S. aureus carriage patterns is the largest in the field, but still we found no association between VDR gene variation and S. aureus nasal carriage.

Notes: Eumycetoma due to Madurella mycetomatis is a major mycological health problem in endemic areas. We infected BALB/c mice (male or female) with various amounts of M. mycetomatis mycelium, containing sterilized soil as a natural adjuvant or Freund's incomplete adjuvant. Mice differed with respect to age and immune status. Intraperitoneal, subcutaneous and intravenous inoculation was explored and survival was monitored. Mice were killed at various intervals after inoculation, checked for the presence of the characteristic black grains, and organs were cultured for M. mycetomatis. Infected organs were subjected to histopathological examination. Immunocompetent male mice were as susceptible as immunocompromised female mice, but showed higher mortality rates. In conclusion, a reproducible mouse model of intraperitoneal M. mycetomatis infection with characteristic black grains in immunocompetent adult or young female mice was developed. Although this experimental model does not simulate macroscopic features of the subcutaneous M. mycetomatis infection in humans, the histopathological characteristics of the lesions and the development of black grains are clearly representative for the human infection. This model will enable further studies on the pathogenesis as well as prevention and treatment of the fungal infection.