S1 De voorjaarsbijeenkomst van de Nederlandse ... - NVMM

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collectively referred to as C. difficile associated disease (CDAD). The cell cytotoxicity (CT) assay is considered the gold standard for detection of toxigenic C. difficile. However, this method is laborious and time-consuming (turn-around time: >48 hours). Rapid diagnosis of CDAD is important, since it may result in early treatment and help prevent nosocomial transmission. Therefore, a real-time PCR based assay was developed and validated. Also, this assay will be used as a screening tool for a gastroenteritis study (GEops Study). This study will commence in May 2008 in 6 hospitals in the Netherlands. Its primary goal is to assess the incidence, etiology and course of patients hospitalised for gastroenteritis. Methods: An assay targeting the tcdA and tcdB genes was developed. Stools were processed with the easyMAG specific A stool protocol (bioMérieux). The phocine herpes virus-1 was used as internal control. The selectivity of the assay was validated with a panel of well characterised C. difficile strains and clinical isolates (n=50) and non-C difficile strains (n=43). The analytical sensitivity was assessed by dilution series (n=3), spiked in a homogenous faecal matrix. Also, the assay was compared with the CT assay in a clinical validation performed on stool samples (n=163) of patients suspected of CDAD. Results: The screening assay proved to be specific for C. difficile as no cross-reaction was observed. The assay was capable of detecting approximately 1560 CFU/g of stool with a 100% hit rate. Sixteen PCR positive and 139 PCR negative samples were in complete concordance with the CT assay. The PCR was positive in 8 additional samples which remained negative in the CT assay. One of these 8 samples could be confirmed as truly positive by CT in a consecutive sample of the same patient. In comparison with the gold standard (CT) PCR showed 100% sensitivity, 95% specificity, 67% positive predictive value, and 100% negative predictive value. PCR inhibition was observed in less than 1% of all 163 screened stool samples. Conclusions: 1. This in-house real-time PCR assay offers a rapid and sensitive method for the first screening for CDAD. 2. The assay will be used as a rapid screening tool for the detection of C. difficile in the GEops Study that will commence in May 2008. O061 Characterization of Clostridium difficile ribotype 078 from human and animal origin D. Bakker Dept. KML, Leiden University Medical Centre, Leiden Background: A recent study demonstrates that Clostridium difficile PCR-ribotype 078 (Type 078) is the predominant ribotype in cattle. Type 078 seems to be an emerging Ned Tijdschr Med Microbiol 2008;16:Supplement S26 type in human disease, with an increase from 5.6% of all submitted isolates to the national reference laboratory in 2006 to 10% in 2007. To characterise human and animal type 078 isolates in more detail, we applied ‘multi locus variable tandem repeat analysis’ (MLVA) and investigated the isolates for the presence of resistance genes (ermB) and genes encoding enterotoxin A (tcdA), cytotoxin B (tcdB) and binary toxins (cdtA, cdtB). Additionally, we sequenced a part of the negative regulator (tcdC) of the pathogenesis locus and determined the susceptibility pattern of 51 C. difficile both human (n=43) and pig (n=8) type 078 isolates. The isolates originated from human diarrhoeal patients (n=60) from the year 2005, 2006 and 2007 and from diarrhoeal pigs (n=10). Methods: The presence of the toxin and ermB genes was established by conventional PCR using previously described primers. Sequencing of the tcdC gene was performed by cycle sequencing and analysis. Susceptibility to moxifloxacin, ciprofloxacin, clindamycin and erythromycin was investigated using E-test method using the most recent CLSI breakpoints (2007). MLVA was applied on 19 human and 10 pig isolates. Genetic relationships were determined by clustering isolates according to MLVA type using the summed absolute distance as the coefficient for calculating the minimum-spanning tree. Results: All 51 strains tested positive for tcdA, tcdB, cdtA and cdtB. The ermB gene was present in 50% of pig and 7% of human isolates (average percentage 14%). All 51 characterised isolates contained two point mutations at location 183 bp and 184 bp and had a 39 bp deletion in the tcdC gene. Resistance for moxifloxacin, ciprofloxacin, clindamycin and erythromycin was found in 12, 88, 6 and 75% of the isolates, respectively. MLVA revealed that, with the exception of one human isolate, all (97%) tested human and pig type 078 isolates were genetically related (single locus variants (SLV’s) with a summed tandem repeat difference (STRD) ≤10). Two genetically highly related (clonal) clusters were found (SLV’s with a STRD ≤2), containing both human (n=7) and pig (n=6) isolates. Conclusions: 3. Toxin profiles (tcdA, tcdB, cdtA and cdtB) of human and pig isolates were identical; 4. Most of the isolates (66%) highly resistant to clindamycin and erythromycin are ermB positive. 5. The point mutation at location 184 bp introduces a stopcodon in the tcdC gene leading to a non-functional tcdC gene. 6. In total, 97% of human and pig type 078 isolates were genetically related. Two genetically highly related (clonal) clusters containing both human and pig isolates suggesting zoonotic transmission. 7. C. difficile type 078 isolates have the same virulence markers as the highly virulent C. difficile ribotype 027.
O062 Implications of HPV testing in cervical screening P.J.F. Snijders 1 , D. Heideman 1 , F.J. van Kemenade 1 , H. Berkhof2 , C.J.L.M. Meijer1 1 2 Depts. of Pathology and Clinical Epidemiology and Biostatistics, VU University Medical Centre, Amsterdam In the Dutch organised cervical screening program 750,000 women between 30 and 60 years of age are invited annually for a Pap smear (with a 5 year screening interval). Almost 480,000 of them attend. Two percent of them will receive a repeat advice and 0.8% a referral advice for colposcopy. Current drawbacks are substantial numbers of false negative (an estimated 3,500 CIN2+ lesions remain undetected by cytology) and false positive smears (11,000 women have repeated cytology or are referred to detect 4,500 CIN2+ lesions). Moreover, half of the cervical cancers detected in the 30-60 age group involve women who did not have a prior screening history and the estimation is that 4,500 CIN2+ lesions remain undetected in the 20-25% of women that do not respond to the invitation for screening (i.e. non-responders). We investigated whether high-risk HPV (hrHPV) testing can offer improvement in any of these problematic areas. Firstly, a prospective, randomised trial was initiated to evaluate the effectiveness of hrHPV testing as an adjunct screening tool in a large scale population-based primary screening setting. This POBASCAM (Population based screening Amsterdam) trial compares the yield of CIN3+ among 44,102 women in a primary screening program by either cytology testing alone (control arm) or cytology and HPV testing (intervention arm), using a GP5+/6+ PCR enzyme immunoassay (EIA). Interim analyses up to 18 months of follow-up revealed that the sensitivity and negative predictive value of HPV testing for CIN3+ is superior to that of cytology. Analysis of about 17,000 women who reached already the second screening round (after 5 years) revealed that the number of CIN3+ lesions detected during the baseline round was substantially higher in the intervention than in the control group, although the total number of CIN3+ over two screening rounds did not differ between groups. Therefore, hrHPV testing leads to earlier detection of clinically relevant CIN3+, permitting extension of the screening interval. We also performed a study on non-responder women. Aiming at improving the coverage 2,546 of these women were offered a package for taking self-sampled vaginal specimens at home to be sent to the laboratory for hrHPV testing. Another 284 non-responder women received instead an extra recall for cytology (control group). Active response was higher in the self-sampling than in the control group (34.2% vs 17.6%; p
Page 1 and 2: De voorjaarsbijeenkomst van de Nede
Page 3 and 4: dates 31 March, 1 & 2 April 2008 Ve
Page 5 and 6: FLOORPLAN CONgReSS CeNTRe Ned Tijds
Page 7 and 8: MONdAY MARCH 31 2008 Room Sydney 12
Page 9 and 10: 16:15 - 16:30 A. Goorhuis O057 16:3
Page 11 and 12: 10:15 - 10:30 A.C. Fluit The distin
Page 13 and 14: Room Athene Parallel session ‘The
Page 15 and 16: O011 Oefening baart kunst A. Jacobi
Page 17 and 18: categories, but were enriched for t
Page 19 and 20: organism may influence each others
Page 21 and 22: sequence typing (MLST). In 2005 and
Page 23 and 24: 2005. Ribotype 027 caused new outbr
Page 25: almost weekly in newly born piglets
Page 29 and 30: A significant stress encountered by
Page 31 and 32: Methods: We developed an oligonucle
Page 33 and 34: The two major, clinically relevant
Page 35 and 36: that propagation of these cells doe
Page 37 and 38: case reports and two methodological
Page 39 and 40: to non-compliance with infection co
Page 41 and 42: lethal infection with respiratory v
Page 43 and 44: Patients and methods: In this prosp
Page 45 and 46: the variant secretome seems to cont
Page 47 and 48: - Fraser JA, Giles SS, Wenink EC, G
Page 49 and 50: 7%), and mucus in stool (7% versus
Page 51 and 52: increasing the diagnostic yield in
Page 53 and 54: the complement fixation test (CFT)
Page 55 and 56: animal model for OM and subsequent
Page 57 and 58: O149 Response of Bacillus cereus AT
Page 59 and 60: TEM-52 variant was the most common
Page 61 and 62: second half of 2007 a database was
Page 63 and 64: Onchocerca volvulus The microfilari
Page 65 and 66: Environmental investigations, done
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Page 69 and 70: O183 Regulation of nitrogen metabol
Page 71 and 72: Methods: 252 B. pertussis clinical
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P05 Staphylococcus aureus prevalenc
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2. Antibiotic resistance and mecA c
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lood. The BacTec 9240 system detect
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isk of transfusion. The prevalence
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Pure instrument. These results sugg
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the 16s and the 23s rDNA and specif
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assays. 11 samples were derived fro
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and daunomycin were established. Th
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Burkholderia species, whereas strai
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strains are genetically related to
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did not have overlapping community
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The gene expression level in differ
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P52 Age-related genotypic and pheno
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AMAN cases in January to March. Fif
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specimens. Growth followed by speci
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S. suis is strongly encapsulated, 3
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P68 differential responses of perio
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2. Atkinson RL, Dhurandhar NV, Alli
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Aalten, D.M.F. van O035 Aarle, I. v
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He, Q. O070 Heck, M.E.O.C. O055, P5
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Savage, N. O113 Savelkoul, P.H.M. O
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Colofon Congresnummer Nederlands Ti
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