A dynamic case definition is warranted for adequate notification in an extended epidemic setting: the Dutch Q fever outbreak 2007-2009 as exemplar.

Q fever is a notifiable disease in the Netherlands:laboratories are obliged to notify possible cases to the Municipal Health Services. These services then try to reconfirm cases with additional clinical and epidemiological data and provide anonymised reports to the national case register of notifiable diseases. Since the start of the 2007­2009 Dutch Q fever outbreak,notification rules remained unchanged, despite new laboratory insights and altered epidemiology. In this study, we retrospectively analysed how these changes influenced the proportion of laboratory-defined acute Q fever cases (confirmed, probable and possible)that were included in the national case register, during(2009) and after the outbreak (2010 and 2011).The number of laboratory-defined cases notified to the Municipal Health Services was 377 in 2009, 96 in 2010 and 50 in 2011. Of these, 186 (49.3%) in 2009, 12(12.5%) in 2010 and 9 (18.0%) in 2011 were confirmed as acute infection by laboratory interpretation. The proportion of laboratory-defined acute Q fever cases that was reconfirmed by the Municipal Health Services and that were included in the national case register decreased from 90% in 2009, to 22% and 24% in 2010 and 2011, respectively. The decrease was observed in all categories of cases, including those considered to be confirmed by laboratory criteria. Continued use ofa pre-outbreak case definition led to over-reporting of cases to the Municipal Health Services in the post-epidemic years. Therefore we recommend dynamic laboratory notification rules, by reviewing case definitions periodically in an ongoing epidemic, as in the Dutch Q fever outbreak.

Targeted screening as a tool for the early detection of chronic Q fever patients after a large outbreak.

In the aftermath of the Dutch Q fever outbreak, an increasing number of patients are being diagnosed with chronic Q fever. Most of these patients are unaware of being infected with Coxiella burnetii, the causative agent of Q fever. To find patients in an earlier, asymptomatic stage, a targeted screening strategy (TSS) for patients with risk factors for chronic Q fever was started in the southeast region of Noord-Brabant. In total, 763 patients were tested using an IgG phase II indirect fluorescent antibody test (IFAT), of which 52 (7 %) patients tested positive. Ten of these 52 patients displayed a chronic Q fever serological profile. All of these 10 patients had a heart valve(s) or (endo-)vascular prosthesis. All except one were asymptomatic. Suggestive signs for chronic infections on positron emission tomography-computed tomography (PET-CT) were demonstrated in 5 (50 %) of these patients. Forty-two out of the 52 patients with a positive screening test showed a past Q fever serological profile. After a year of follow-up (every 3 months), none of these patients showed elevation of antibody titres and no new chronic Q fever patients were found in this group. A targeted screening programme is a useful instrument for detecting patients at risk of developing chronic Q fever.

Erythromycin resistance in the commensal throat flora of patients visiting the general practitioner: a reservoir for resistance genes for potential pathogenic bacteria.

The prevalence and mechanism of erythromycin resistance in commensal throat streptococci was determined from October 2000 until December 2002 as part of an ongoing study of the NIVEL in general practice patients (N=678). Resistance prevalence for 1mg/L and 16 mg/L erythromycin was 57% and 20%, respectively. The percentage of total commensal flora resistant within each patient ranged from 1% to 100% (median, 1%). mefA was predominantly found among isolates on the 1mg/L plates, and ermB was found in 64% of the isolates on the 16 mg/L plates. Erythromycin resistance was transferred from a commensal isolate to Streptococcus pneumoniae with a frequency of 1 x 10(-9). Commensal streptococci of general practice patients in The Netherlands form a large reservoir of transferable erythromycin resistance (genes) for potential pathogenic microorganisms.

Prevalence of macrolide resistance genes in clinical isolates of the Streptococcus anginosus ("S. milleri") group.

Twenty-two unrelated erythromycin-resistant anginosus group strains (3.2% resistance rate) were assessed for mechanisms of resistance. Streptococcus anginosus accounted for 16 of the 22 isolates. Fifteen isolates harbored the erm(B) gene. The erm(TR) and the mef(E) genes were carried by two isolates each. In three isolates, none of these resistance genes was detected by PCR.

Misidentification and diagnostic delay Caused by a false-positive amplified Mycobacterium tuberculosis direct test in an immunocompetent patient with a Mycobacterium celatum infection.

The Gen-Probe amplified Mycobacterium tuberculosis direct test can give discrepant results directly in respiratory or cultured samples from patients infected with Mycobacterium celatum, leading to inappropriate therapy for, in our case, an immunocompetent patient.

Chlamydia pneumoniae and Mycoplasma pneumoniae in children with acute respiratory infection in general practices in The Netherlands.

In this retrospective study Chlamydia pneumoniae and Mycoplasma pneumoniae infections were detected by polymerase chain reaction (PCR) in samples (n = 457) from children presenting with acute respiratory infection to general practitioners during 1992-97. Samples were collected in autumn and winter, and from 1994 onwards in spring and summer also. Overall, C. pneumoniae and M. pneumoniae were detected in throat or nasal samples by PCR in 3.1% and 2.4% of the cases, respectively. The proportion of both C. pneumoniae and M. pneumoniae infections varied between 0% and 6.9% over the years studied, whereas seasonal proportions varied from 1.8 to 9.1% and 1.2 to 4.5%, respectively. For both microorganisms the lowest proportion was detected during winter and the highest in summer. C. pneumoniae could already be detected by PCR in patients under 4 y of age, an observation not made in sero-epidemiological studies. In conclusion, both C. pneumoniae and M. pneumoniae infections play a minor role in children presenting with acute respiratory infection.

Application of different genotyping methods for Pseudomonas aeruginosa in a setting of endemicity in an intensive care unit.

Colonization with Pseudomonas aeruginosa was studied by taking serial swab specimens from the oropharynges and anuses and tracheal and gastric aspirates from patients in an intensive care unit during a 10-month period in a setting of endemicity. Nineteen (10%) of the 192 patients included in the study were colonized on admission, while another 30 (16%) patients acquired P. aeruginosa while in the hospital. Typing of 353 isolates was performed by random amplified polymorphic DNA (RAPD) analysis, and 56 strains were selected for further typing by RAPD analysis, pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) analysis. By these methods, 42, 44, and 44 genotypes were found, respectively. Computer-aided cluster analysis indicated that similar groups of related isolates were obtained by each method. By taking admission periods into account, analysis of the typing results suggested cross-acquisition of P. aeruginosa for five patient pairs. The small number of transfers and the large number of genotypes found indicate that most P. aeruginosa strains were derived from the patients themselves. The numbers of observed typing patterns and band differences between related isolates were counted for each typing method. AFLP analysis with primers without a selective base proved to be the most discriminatory method, followed by PFGE, AFLP analysis (with one selective base), and RAPD analysis. On the basis of a comparison with established strain differentiation criteria for PFGE, the criteria for differentiation of P. aeruginosa by AFLP analysis are presented.

Improvement of growth of Chlamydia pneumoniae on HEp-2 cells by pretreatment with polyethylene glycol in combination with additional centrifugation and extension of culture time.

The following adaptations led to improved growth of Chlamydia pneumoniae on HEp-2 cells compared to that by the standard method: monolayer preincubation with 7% polyethylene glycol (PEG), extension of incubation time to 7 days, and extension of incubation to 7 days in combination with centrifugation on days 3, 4, and 5. These adaptations resulted in approximate increases in numbers of inclusion-forming units (IFU) of 2-, 5-, and 69-fold, respectively. A combination of preincubation with PEG, prolonged incubation, and centrifugation on days 3, 4, and 5 increased the numbers of IFU >300-fold. This is therefore recommended as the optimal method for culturing C. pneumoniae.

Fatal encephalitis caused by Mycoplasma pneumoniae in a 9-year-old girl.

A case of fatal encephalitis in a 9-year-old girl is described. Serology showed high titre antibodies against Mycoplasma pneumoniae. In addition M. pneumoniae was detected in cerebrospinal fluid by polymerase chain reaction. Direct invasion of the central nervous system as opposed to a secondary immunologic reaction to a M. pneumoniae infection of the respiratory tract in the pathogenesis of encephalitis is discussed.

Delta APACHE II for predicting course and outcome of nosocomial Staphylococcus aureus bacteremia and its relation to host defense.

To predict at an early phase the clinical course and outcome of nosocomial Staphylococcus aureus bacteremia, the APACHE II score at onset of bacteremia was calculated in 99 patients. A delta APACHE II score (i.e., the difference between this score and one calculated for the day before the bacteremia) was also determined. This delta APACHE II score was highly significantly correlated with clinical course (P<.001) and outcome (P<.001). The risk of a complicated clinical course and of dying from S. aureus bacteremia is determined at the very onset of bacteremia, and this risk can best be assessed by calculating the delta APACHE II score. Furthermore, a positive correlation was found between delta APACHE II scores and the level of serum opsonic activity (P=.003) toward S. aureus. Therefore, the complicated clinical course of S. aureus bacteremia is not due to a relative lack of specific opsonins.

Direct PCR enables detection of Mycoplasma pneumoniae in patients with respiratory tract infections.

The sensitivities of three methods of detection of Mycoplasma pneumoniae by a 16S rDNA PCR were compared by using a serial dilution of M. pneumoniae. These methods consisted of a PCR performed directly on cells after a proteinase K pretreatment (direct PCR), a PCR after purification of nucleic acids (DNA-PCR), and a PCR with rRNA sequences as the target after reverse transcription. The direct PCR and the reverse transcription PCR had a sensitivity of 1.5 CFU (approximately 250 genomes). By purification, a 10-fold loss of target DNA occurred, as shown by a 10-fold decrease in sensitivity (15 CFU) of the DNA-PCR. The presence of an excess of human background DNA did not influence the sensitivity of either PCR. The direct PCR was evaluated on samples from patients with respiratory complaints. Direct PCR amplification was possible in 94.9% of the samples, which were tested by amplification of a 326-bp fragment of the beta-globin gene, which was performed to test for the suitability of amplification. Nucleic acid purification was performed on the beta-globin-negative samples, after which only 2% remained negative. A positive correlation between the direct M. pneumoniae PCR and serology, as tested by the microparticle agglutination assay (MAG assay), was found in 88.1% of the cases. A positive MAG assay result was found for samples from 10 (17%) of the patients; samples from 6 (10.2%) of these patients were also positive by PCR. Samples from three patients were found to be positive by the M. pneumoniae PCR and negative by the MAG assay. Persistence of M. pneumoniae, as detected by PCR was observed in three patients. These results indicate that the direct PCR with 16S rDNA could prove to be useful in the detection of M. pneumoniae in respiratory tract samples, although more studies are needed to evaluate the correlation between clinical symptoms and positive test results.