trialregister.nlIdentifier: NTR1780.

A typing procedure for methicillin-resistant Staphylococcus aureus (MRSA) based on the polymerase chain reaction (PCR) amplification of both mecA sequences and variable DNA sequences as present in the prokaryotic genome has been developed. Two primers based on the sequences of DNA repeats as discovered in gram-negative members of the family Enterobacteriaceae allow detection of variable regions in the genome of a gram-positive bacterium such as S. aureus, as does a newly described arbitrary primer. This procedure, enabling the detection of 23 different genotypes in a collection of 48 MRSA isolates, was validated by comparisons with phage typing studies. It appeared that within the same group of isolates only 13 different phagovars could be identified. Combination of the results from both phage typing and genotyping allowed the discrimination of 34 of 48 isolates. However, depending on the primer-variable complexity of the PCR fingerprints, which could also be modulated by combination of PCR primers, clear homologies between the groups defined by either phage typing or fingerprinting were observed. An analysis of an MRSA outbreak in a geriatric institution showed a collection of genetically homogeneous isolates. In agreement with phage typing, PCR fingerprinting revealed the identical natures of the MRSA strains isolated from all patients.

In 1999, Engelen and coworkers investigated colonization in Amsterdam among 259 children attending 16 day-care centers (DCCs) and among 276 children who did not attend day-care centers (NDCCs). A 1.6-to 3.4-fold increased risk for nasopharyngeal colonization was observed in children attending DCCs compared with NDCC children, while no difference in antibiotic resistance was found between groups. The serotype and genotype distributions of 305 nasopharyngeal Streptococcus pneumoniae isolates of the latter study were investigated. The predominant serotypes in both the DCC and the NDCC groups included 19F (19 and 18%, respectively), 6B (14 and 16%, respectively), 6A (13 and 7%, respectively), 23F (9 and 7%, respectively), and 9V (7 and 7%, respectively). The theoretical vaccine coverage of the 7-valent conjugate vaccine was 59% for the DCC children and 56% for the NDCC group. Genetic analysis of the pneumococcal isolates revealed 75% clustering among pneumococci isolated from DCC attendees versus 50% among the NDCC children. The average pneumococcal cluster size in the DCC group was 3.8 and 4.6 isolates for two respective sample dates (range, 2 to 13 isolates per cluster), while the average cluster size for the NDCC group was 3.0 (range, 2 to 6 isolates per cluster). Similar to observations made in other countries, these results indicate a higher risk for horizontal spread of pneumococci in Dutch DCCs than in the general population. This study emphasizes the importance of molecular epidemiological monitoring before, during, and after implementation of pneumococcal conjugate vaccination in national vaccination programs for children.

Nasopharyngeal carriage of Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis was studied in 259 children attending day care centers (DCC) in Amsterdam, The Netherlands, and in 276 control children. The DCC children were sampled a second time after 4 weeks. Carriage rates for DCC children and controls were 58 and 37% for S. pneumoniae, 37 and 11% for H. influenzae, and 80 and 48% for M. catarrhalis, respectively. No increased antibiotic resistance rates were found in strains isolated from DCC children. All H. influenzae isolates were typed by random amplified polymorphic DNA (RAPD) analysis. Evidence for frequent transmission of H. influenzae strains within DCC was found. In the control group only two isolates (4%) displayed identical RAPD types versus 38% of strains from DCC children. Colonization with H. influenzae appeared to be short-lived in these children; more than half of the children harboring H. influenzae in the first sample were negative in the second sample, whereas most children still positive in the second sample had a different genotype than in the first sample. Of the newly acquired strains in the second sample, 40% were identical to a strain that had been found in a child in the same DCC in the first sample. DCC are to be considered epidemiological niches with a high potential for the spread of pathogenic microorganisms.

The most common etiologic agents of genital ulcer disease (GUD) are herpes simplex virus type 1 (HSV-1), HSV-2, Treponema pallidum, and Haemophilus ducreyi. In an outpatient clinic for sexually transmitted diseases in Amsterdam, The Netherlands, specimens from 372 patients with GUD were collected from February to November 1996. Sera were collected at the time of the symptoms and, for most patients, also during follow-up visits. Swabs in viral transport medium were used for HSV culture and for detection of DNA. The most prevalent pathogen found was HSV-2, which was detected by culture in 35% of the patients and by PCR in 48% of the patients. Also, HSV-1 infection was more often detected by PCR (7.8%) than by culture (5.6%). Evidence for an active infection with T. pallidum was found in 1.9% of the patients, using serological tests. A multiplex PCR for simultaneous T. pallidum and H. ducreyi DNA detection was positive for T. pallidum in 3.3% of the samples and for H. ducreyi in only 0.9% (3 out of 368) of the samples. The sensitivity of the PCR was superior to that of culture for HSV detection and to that of serology for T. pallidum detection. Specific H. ducreyi immunoglobulin G antibodies were detected in sera of 5.2% of the patients, with no concordance between serology and PCR. In 37% of the cases, none of the tested microorganisms was detected. Performance of PCR in addition to conventional techniques significantly improved the diagnosis of GUD.