Margaretha Ieven

Third Belgian multicentre survey of antibiotic susceptibility of anaerobic bacteria.

OBJECTIVESnTo collect recent data on the susceptibility of anaerobes and to compare them with results from previous studies.nnnMETHODSnFour hundred and forty-three anaerobic clinical isolates from various body sites were prospectively collected from October 2003 to February 2005 in nine Belgian hospitals. MICs were determined for nine anti-anaerobic and three recently developed antibiotics.nnnRESULTSnMost gram-negative bacilli except Fusobacterium spp. were resistant to penicillin. Piperacillin/tazobactam, metronidazole, chloramphenicol, meropenem and amoxicillin/clavulanic acid were very active against all groups, but only 86% of Bacteroides fragilis group strains were susceptible to the latter. Cefoxitin, cefotetan and clindamycin were less active. In particular, only 62%, 52% and 48% of B. fragilis group strains were susceptible, respectively. Clindamycin shows a continuing decrease in activity, as 83% were still susceptible in 1987 and 66% in 1993-94. Anti-anaerobic activity of the new antibiotics is interesting, with MIC50 and MIC90 of 1 and >32 mg/L for moxifloxacin, 2 and 4 mg/L for linezolid and 0.5 and 8 mg/L for tigecycline.nnnCONCLUSIONSnThe susceptibility of anaerobic bacteria remains stable in Belgium, except for clindamycin, which shows a continuous decrease in activity. However, for each of the tested antibiotics, at least a few resistant organisms were detected. Consequently, for severe infections involving anaerobic bacteria, it could be advisable to perform microbiological testing instead of relying on known susceptibility profiles. Periodically monitoring background susceptibility remains necessary to guide empirical therapy.

Evaluation of NucliSens easyMAG for automated nucleic acid extraction from various clinical specimens.

ABSTRACT The objectives of this study were to evaluate the performance of the NucliSens easyMAG platform for nucleic acid extraction from different clinical specimens compared to NucliSens miniMAG platform and manual QIAGEN extraction. The NucliSens easyMAG and the NucliSens miniMAG showed equal performance on 215 throat swabs since real-time nucleic acid sequence-based amplification scored the same samples positive for Mycoplasma pneumoniae (n = 9) and Chlamydia pneumoniae (n = 5) RNAs, although internal control RNA was slightly better detected with the NucliSens easyMAG (99.3% versus 96.8%). NucliSens easyMAG extracted nucleic acids more efficiently (higher recovery and/or fewer inhibitors) compared to QIAGEN extraction by showing, on average, lower Ct values in real-time LightCycler PCR, although 4 individual specimen out of 45 were found positive only with QIAGEN. For nine M. pneumoniae-positive throat swabs, the mean difference in Ct values between NucliSens easyMAG extraction and QIAGEN extraction was −2.26 (range, −5.77 to +0.60); for the detection of five C. pneumoniae-positive throat swabs, the average difference in Ct values between the two methods was −3.38 (range, −6.62 to −2.02); and for the detection of cytomegalovirus in 24 blood samples, the mean difference in Ct values between the two methods was −0.95 (range, −5.51 to +1.68). The NucliSens easyMAG is considerably easier to perform, efficiently extracts nucleic acids from throat swabs and whole blood, is automated, and has high throughput.

Detection of Mycoplasma pneumoniae by Real-Time Nucleic Acid Sequence-Based Amplification

ABSTRACT Real-time isothermal nucleic acid sequence-based amplification (RT-NASBA) was applied to the detection of Mycoplasma pneumoniae. In vitro-generated M. pneumoniae RNA was used to assess the sensitivity of the assay. The 95% hit rate was 148 molecules of M. pneumoniae RNA in the amplification and 104 molecules of in vitro-generated RNA after nucleic acid extraction. The sensitivity of the RT-NASBA and the conventional NASBA assays corresponded to 5 color-changing units (CCU) of M. pneumoniae. In spiked throat swabs, nasopharyngeal aspirates, bronchoalveolar lavages, and sputum, the sensitivity of both NASBA assays corresponded to 5 to 50 CCU of M. pneumoniae. A total of 17 clinical specimens positive for M. pneumoniae by PCR were also positive by conventional NASBA, but one specimen was negative by RT-NASBA. These results indicate that the sensitivity of detection of M. pneumoniae by RT-NASBA in respiratory samples might be slightly reduced compared to that by conventional NASBA. However, the real-time assay is superior in speed and ease of handling.

Prevalence and Molecular Epidemiology of Glycopeptide-Resistant Enterococci in Belgian Renal Dialysis Units

The molecular epidemiology of glycopeptide-resistant enterococci (GRE) colonizing the intestinal tracts of Belgian renal dialysis patients was studied among 1318 patients of a population of 1800 dialysis patients from 29 dialysis centers. Of these, 185 patients (14.0%) were colonized with a VANA-positive GRE; GRE harboring the VANB gene were not detected. The majority of the VANA GRE (80.5%) were identified as Enterococcus faecium; 14.8% were identified as E. faecalis; and a limited number were identified as E. avium, E. casseliflavus, E. dispar, E. durans, or E. gallinarum. Genome analysis of 277 VANA-positive GRE by pulsed-field gel electrophoresis revealed a high genetic variability both within the different dialysis centers and within the patients own GRE flora. No high-level gentamicin-resistant VANA-positive GRE were detected, and most strains remained susceptible to ampicillin. These findings do not support a hospital-driven endemicity of VANA-positive enterococcal isolates in Belgium.

Relationship between histologic chorioamnionitis and early inflammatory variables in blood, tracheal aspirates, and endotracheal colonization in preterm infants

Histologic results of the placenta are usually not available within the first days of life. We identified inflammatory variables in tracheal aspirates and blood that were associated with histologic chorioamnionitis (HC). A derivation cohort consisted of 62 neonates and a validation cohort of 57 neonates with a gestational age < 31 wk and ventilated on d 1. Tracheal aspirates were taken on d 1 and on d 3, if the patient was still ventilated. HC was diagnosed by light microscopy. Logistic regression was used to identify independent factors in the derivation cohort associated with HC at d 1, 2, and 3. Model performance was studied using receiver operating characteristic curve analysis. Independent factors associated with HC were, at d 1, tracheal aspirate IL-8 ≥ 917 pg/mL (odds ratio, 60.7; 95% confidence interval, 11-328); at d 2, blood C-reactive protein ≥ 14 mg/L (odds ratio, 9.2; 95% confidence interval, 2-38), blood white blood cell count ≥ 10,400/mm3 (odds ratio, 7.4; 95% confidence interval, 2-28); and at d 3, blood neutrophil count ≥ 4968/mm3 (odds ratio, 14; 95% confidence interval, 3-57). The association with HC was less at d 3 (area under receiver operating characteristic curve, 0.77) when compared with the d 1 model (area under the curve, 0.88; p = 0.09). The models performed equally well in the validation cohort (goodness-of-fit test, p > 0.05). We conclude that the d 1 and d 2 models can be used as diagnostic factors for HC. Tracheal aspirate IL-8 taken immediately after birth was equally accurate in the diagnosis of HC as systemic inflammatory response at d 2 and better than on d 3.

Development of Conventional and Real-Time Nucleic Acid Sequence-Based Amplification Assays for Detection of Chlamydophila pneumoniae in Respiratory Specimens

ABSTRACT Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Chlamydophila pneumoniae 16S rRNA by using the NucliSens basic kit (bioMérieux, Boxtel, The Netherlands). The assay was originally developed as a conventional NASBA assay with electrochemiluminescence detection and was subsequently adapted to a real-time NASBA format by using a molecular beacon. C. pneumoniae RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild-type C. pneumoniae RNA and 0.1 inclusion-forming unit (IFU) of C. pneumoniae. In spiked respiratory specimens, the sensitivity of the C. pneumoniae NASBA assay varied between 0.1 and 1 IFU/100 μl sample, depending on the type of specimen. Finally, conventional and real-time NASBA were applied to respiratory specimens previously tested by PCR. A 100% concordance between the test results was obtained.

Predicting the presence of bacterial pathogens in the airways of primary care patients with acute cough

BACKGROUND: Bacterial testing of all patients who present with acute cough is not feasible in primary care. Furthermore, the extent to which easily obtainable clinical information predicts bacterial infection is unknown. We evaluated the diagnostic value of clinical examination and testing for C-reactive protein and procalcitonin for bacterial lower respiratory tract infection. METHODS: Through a European diagnostic study, we recruited 3104 adults with acute cough (≤ 28 days) in primary care settings. All of the patients underwent clinical examination, measurement of C-reactive protein and procalcitonin in blood, and chest radiography. Bacterial infection was determined by conventional culture, polymerase chain reaction and serology, and positive results were defined by the presence of Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Bordetella pertussis or Legionella pneumophila. Using multivariable regression analysis, we examined the association of diagnostic variables with the presence of bacterial infection. RESULTS: Overall, 539 patients (17%) had bacterial lower respiratory tract infection, and 38 (1%) had bacterial pneumonia. The only item with diagnostic value for lower respiratory tract infection was discoloured sputum (area under the receiver operating characteristic [ROC] curve 0.56, 95% confidence interval [CI] 0.54–0.59). Adding C-reactive protein above 30 mg/L increased the area under the ROC curve to 0.62 (95% CI 0.59–0.65). For bacterial pneumonia, comorbidity, fever and crackles on auscultation had diagnostic value (area under ROC curve 0.68, 95% CI 0.58–0.77). Adding C-reactive protein above 30 mg/L increased the area under the ROC curve to 0.79 (95% CI 0.71–0.87). Procalcitonin did not add diagnostic information for any bacterial lower respiratory tract infection, including bacterial pneumonia. INTERPRETATION: In adults presenting with acute lower respiratory tract infection, signs, symptoms and C-reactive protein showed diagnostic value for a bacterial cause. However, the ability of these diagnostic indicators to exclude a bacterial cause was limited. Procalcitonin added no clinically relevant information.

Decreasing incidence of neonatal nosocomial bloodstream infections in a neonatal intensive care unit: antenatal corticosteroid treatment an innocent bystander?

We studied the effect of the use of antenatal steroid treatment on the incidence of nosocomial bloodstream infections (NBSI). All episodes of culture proven NBSI occurring after 96xa0h of hospitalisation were identified retrospectively during a 10-year period (1991–2001). Throughout the study period, the use of antenatal steroids, demographic characteristics and morbidity of the patients were recorded prospectively. Since 1996 more efforts were made to use antenatal steroids to decrease neonatal morbidity and mortality. The incidence rates of NBSI were compared between period 1 (1991–1995) and period 2 (1996–2001). The overall incidence rate of NBSI dropped significantly from 7.4% (6.1%–8.9%) in period 1 to 5.0% (4.0%–6.2%) in period 2 and was most pronounced in the birth weight category 1000xa0g–1500xa0g (11.7%, 7.9%–15.0% to 6.9%, 4.3%–10.5%) and 1500xa0g–2500xa0g (3.6%, 2.2%–5.6% to 1.4%, 0.6%–2.8%). Antenatal use of steroids increased overall from 19% in 1991 to 51% in 2001 (P<0.001). Since 1996 there was a decreasing number of ventilation days (P=0.011) and decreasing incidence of patent ductus arteriosus (P=0.001), while the incidence of neonatal surgery, chronic lung disease and duration of hospitalisation remained constant over time. Conclusion: increased use of antenatal steroids is associated with a decreasing incidence rate of nosocomial bloodstream infections in neonates with birth weights between 1000xa0g and 2500xa0g, probably by decreasing the incidence of patent ductus arteriosus and/or due to improved respiratory outcome. This finding needs to be confirmed by randomised control trials or by a large prospective cohort study in similar population groups.

Organization of the Ribosomal Operon 16S-23S Gene Spacer Region in Representatives of Neisseria gonorrhoeae

Ribosomal rRNA gene fragments (rDNA) encompassing part of the 16S rDNA, the 16S-23S rDNA spacer region and part of the 23S rDNA of 229 Neisseria gonorrhoeae strains were enzymatically amplified using conserved primers. The fragments of approximately 1200 bp were subjected to restriction analysis with HinfI. This revealed 13 patterns (patterns I-XIII) of which patterns I (78 strains), II (32 strains), III (38 strains) and IV (56 strains) were the most abundant, comprising 89.1% of the strains. The obtained restriction patterns consisted of 3 to 8 bands, ranging in size from 32 to 854 bp. The sum of the obtained bands was about 1200 bp for patterns I, II, III, IV, V, IX, and XIII. However, for patterns VI, VII, VIII, X, XI and XII, the sum of the bands well exceeded the estimated size of approximately 1200 bp. We demonstrated that this results from sequence divergence in the 4 rRNA operons, present in the genome of N. gonorrhoeae, giving rise to patterns that are a combination of several other patterns.

Sepsis Caused by Achromobacter Xylosoxidans in a Child with Cystic Fibrosis and Severe Lung Disease

Patient: Female, 10 Final Diagnosis: Sepsis Symptoms: Fever • hypotension • not tollerating enteral feeds • respiratory deterioration Medication: — Clinical Procedure: IV antibiotics • lungtransplantion Specialty: Pediatrics and Neonatology Objective: Unusual clinical course Background: Achromobacter xylosoxidans is an aerobic, motile, Gram-negative, opportunistic pathogen that can be responsible for various severe nosocomial and community-acquired infections. It has been found in immunocompromised patients and patients with several other underlying conditions, but the clinical role of this microorganism in cystic fibrosis is unclear. Case Report: We describe a case of septic shock caused by A. xylosoxidans in a 10-year-old child with cystic fibrosis and severe lung disease. Conclusions: As the prevalence of A. xylosoxidans in cystic fibrosis patients is rising and patient-to-patient transmission is highly probable, further studies are warranted to determine its role and to document the appropriate treatment strategy for eradication and long-term treatment of this organism.