O480 Characterisation of Neisseria meningitidis B causing invasive disease in the Czech Republic

S102 differentiate M. tuberculosis (IS6110 positive – RD9 positive) from the other mycobacterial species included in the M. tuberculosis complex (IS6110 positive – RD9 negative). We recently evaluated the first version of the RT-TB kit, demonstrating that the assay is sensitive, particularly in smear negative–culture positive clinical samples. Here, we report on the evaluation of a new version of the RT-TB assay in which the labelling of the IS6110 probe has been modified in order to improve its stability and detection level. Methods: The new RT-TB BioRad kit includes an IS6110-BRD04 probe (instead of IS6110-Tamra in the previous version) that increases by 10-fold the sensitivity of detection of the IS6110 amplicons. Fifty-two clinical samples collected in routine (sputa, bronchoalveolar and gastric lavages) were included. DNA preparations and RT PCR reactions were carried out according to the manufacturer’s instructions. A new specific software developed by the manufacturer was used for the automated analysis of the RT-TB amplification results. Results: In the present study, 2 samples were found to contain inhibitors (as indicated by the negative amplification signals of the corresponding internal controls). The 8 smear positive samples included in the study were all found to be IS6110-POS and RD9-POS. Interestingly, 4 of them showed <1 acid fast bacilli per field on microscopic examination. The good sensitivity of the test was confirmed by 2 smear negative samples which were both found to be IS6110-POS and RD9-POS by the RT-TB assay. These two samples were confirmed to be positive for M. tuberculosis by the Roche Amplicor assay. Finally, the 40 remaining smear-negative samples were all found to be negative for IS6110 and RD9, suggesting that the increased sensitivity of the new IS6110-BRD04 probe does not impair the specificity of the test. Conclusion: The results obtained for the first evaluation of the new version of the RT-TB kit suggest that the assay is sensitive and specific and may be very promising for the detection of M. tuberculosis in clinical samples containing few bacilli. Further experiments are in progress to confirm these preliminary data. O478 Rapid detection of rifampin resistance mutations in clinical isolates of Mycobacterium tuberculosis by Dot-Blot hybridisation assay M. Varma-Basil, R. Pathak, S. Ahmed, A. Bhatnagar, M. Bose (Delhi, IN) Objectives: A Dot-Blot hybridisation assay that detects all mutations occurring in the M. tuberculosis rpoB hot-spot region is being developed. The assay uses five probes, capable of binding to a different target segment within the rpoB hot-spot region of the wild-type M. tuberculosis genome. The present study is a preliminary investigation to assess the suitability of the assay for detection of resistance mutations in rpoB in clinical isolates of M. tuberculosis from Delhi, India. Methods: Susceptibility testing of 142 isolates of M. tuberculosis was carried out by proportion method and confirmed by BACTEC 460TB system. Dot-Blot assay was performed on 106 isolates with two of the probes hybridising to the wild-type sequence of M. tuberculosis, from codons 522 to 527 (Probe D) and 528 to 533 (Probe E). Absence of hybridisation with any of the probes in the assay when a mutation was present indicated Rifampicin resistance, a surrogate marker for Multidrug-resistant M. tuberculosis. Results: Susceptibility testing of 142 isolates revealed isoniazid resistance in 53.5%, rifampicin resistance in 41%, streptomycin resistance in 53% and ethambutol resistance in 37% of strains. Fortyfive strains (32%) were multidrug resistant. Further analysis of the data showed that 96.4% of rifampicin-resistant strains and 100% of ethambutol-resistant strains had co resistance to one or the other antituberculous drug. It was also interesting to note that 77% of the rifampicin-resistant strains were multidrug resistant, while the corresponding figure for ethambutol-resistant strains was 87%. Dot-Blot hybridisation assay with probes D and E was carried out on 106 isolates of M. tuberculosis, of which 43 were resistant to rifampicin. Of the rifampicin-resistant isolates tested, 15 did not hybridise with probe E, indicating a mutation at this site. The results of 10 strains were confirmed by sequencing when a mutation was detected in 9 strains at codon 531

17th ECCMID / 25th ICC, Oral presentations and in one of them at codon 533. The remaining 28 rifampicin-resistant strains, which hybridised with probe E, may have a mutation at a site other than that complimentary to probe E. Of these, 10 hybridised with probe D, indicating a mutation at the site complimentary to probe D (codons 522 to 527). Of the 63 rifampicin-susceptible isolates, 62 (98%) hybridised with probes D and E, indicating a wild-type sequence. Conclusions: The Dot-Blot assay was found to be a sensitive and specific assay.

O479 Rapid detection of multidrug-resistant and heteroresistant tuberculosis in one day using the new molecular-biological test Genotype MTBDRTM H. Hoffmann, G. Murmusaeva, G. Uzakova, B. Shrestha, I. Bozo, S. Yann, K. Feldmann (Gauting, DE; Tashkent, UZ; Kathmandu, NP; Santa Cruz, BO; Donetzk, UA) MDR-TB is defined by resistance of the respective pathogen (Mycobacterium tuberculosis complex, MBTC) against INH and rifampin RMP. Mortality of MDR-TB is double compared to sensitive TB. Phenotypic drug-susceptibility-testing (DST) lasts up to 8 weeks. Following the DOTS and waiting for DST results, MDR-TB patients are miss-treated with INH and RMP. A new resistance test, Genotype MTBDRTM (G-MDR), has been released, detecting MDR-TB. The test is based on PCR and hybridisation. For its validation, 64 strains have been characterised by threefold DST on solid and liquid media. 100% of RMP resistances (n = 31) and 90% of INH resistances (n = 49) were detected by G-MDR. Specifity was 100%. 112 TB isolates from highly endemic countries were analysed: 31 from Bolivia, 58 from Nepal and 23 from Ukraine. 93% of RMP-resistances were detected with G-MDR. The sensitivity for detection of INH resistance increased with the prevalence of MDR-TB in the respective countries from 64% in Nepal, 74% in Bolivia, 83% in Usbekistan, to 86% in the Ukraine. All G-MDR assays for Nepalese strains were performed in the National Reference TB-Laboratory in Katmandu, demonstrating that the test is robust enough to be applied in developing countries. Finally, the test was applied to 35 sputum specimen from Usbekistan, which were all positive in microscopy. All MDR cases have been detected. Additionally, heteroresistance to INH and RMP could be visualised in nine cases. Genotype MTBDRTM is a fast, easy to perform and robust test, applicable in highly endemic countries. Its sensitivity for the detection of RMP resistance is 100%, for INH up to 90% depending on the MDR prevalence. It is an excellent tool for the detection of heteroresistance. The test can be performed from microscopically positive sputum, reducing the DST time to zero days.

Molecular diagnostics O480 Characterisation of Neisseria meningitidis B causing invasive disease in the Czech Republic P. Kriz, J. Kalmusova, M. Musilek, J. Felsberg, R. Haugvicova, D. Caugant, K. Jolley, M. Maiden (Prague, CZ; Oslo, NO; Oxford, UK) Objectives: Invasive meningococcal disease (IMD) caused by Neisseria meningitidis B is endemic in the Czech Republic and its long-term incidence is stable (0.5/100,000 population), reaching peaks in the youngest age groups (up to 22.6/100,000 for 0−11 months olds and 2.7/100,000 for 1−4 years olds). The aim of this study was to characterise N. meningitidis B isolates from IMD and to assess the coverage of vaccines against N. meningitidis B. Methods: The total number of N. meningitidis B isolated from IMD in the Czech Republic in the period 1993–2006 (Nov. 8) was 474. In all isolates serogrouping and sero/subtyping was performed. Subtyping by Whole Cell ELISA (WCE) was replaced by PorA sequencing recently (http://neisseria.org/nm/typing/). Sequence types (STs) were identified by multilocus sequence typing (MLST) in accordance with the MLST

Molecular diagnostics website (http://pubmlst.org/neisseria/). The number of N. meningitidis B IMD isolates investigated by MLST was 366. Results: Sero/subtyping showed high heterogeneity: among 474 isolates, 88 phenotypes were found. The most frequent phenotype was B:4:P1.15 (15.4%), followed by B:15:P1.7,16 (8.2%) and B:15:P1.5 (5.7%). 15.4% of isolates were not typeable/subtypeable by WCE. PorA sequencing of these isolates further identified their heterogeneity. MLST confirmed this high heterogeneity: 142 STs, belonging to 16 clonal complexes, which represent 79.2% of isolates. There were three prevailing complexes: ST-18 complex (19.1%), ST-32 complex (18.8%) and ST-41/44 complex (16.9%). The Czech N. meningitidis B population is different compared to western Europe: 109 of the STs (76.8% of STs) and one clonal complex (ST-292 complex) were described for the first time in the Czech N. meningitidis B isolates. Coverage of sero/subtypes by currently being developped vaccines against N. meningitidis B is low (maximum 44.5% for nine-valent meningococcal B PorA vaccine). Conclusion: Detailed characterisation of N. meningitidis B isolates from IMD in the Czech Republic in the period 1993–2006 showed their high heterogeneity and low coverage by currently available vaccines against N. meningitidis B. Acknowledgement: This work was supported by Ministry of Health, by research grant 1A/8688−3/2005 of the Internal Grant Agency of Ministry of Health of the Czech Republic and made use of the Multi Locus Sequence Typing website (http://pubmlst.org/neisseria/) sited at the University of Oxford and funded by the Wellcome Trust and European Union.

O481 Detection by DHPLC of 23S rDNA mutations responsible for clarithromycin resistance in Helicobacter pylori C. Coulon, C. Lascols, S. Pissard, L. Deforges, J.C. Delchier, C.J. Soussy, E. Cambau (Cr´eteil, FR) Objectives: Resistance to clarithromycin is the leading cause of decrease in H. pylori eradication. Since 23S rRNA mutations correlated with in vitro and in vivo clarithromycin resistance, routine detection of these mutations will be helpful to adapt the antibiotic treatment. Methods: A total of 123 clinical strains of H. pylori consecutively isolated between 2004 and 2005 in our hospital have been studied. Clarithromycin MICs were determined by both the agar dilution and E-test method. A 147-bp fragment, encompassing the nucleotides involved in clarithromycin resistance at positions 2146 and 2147 in 23S rDNA, were amplified and analysed by denaturing high pressure liquid chromatography (WAVE® systems, Transgenomic). The DHPLC analysis was done on PCR products after mixing PCR product from the tested strain with that from a wild-type susceptible reference strain in order to create heteroduplexes. The 23S rDNA was also studied by direct sequencing for all clarithromycin-resistant strains and a large part of susceptible strains. Results: DHPLC profiles were identical to that of the wild-type strain for 85/123 (69%) strains, which were all susceptible to clarithromycin (MICs from 0.06 to 0.5 mg/l) except two strains for which a mix of susceptible and resistant (MIC of 8 and 256 mg/l) strains was observed. DHPLC profiles were different from that of the wild-type strain for 35 strains (28.5%) which were all resistant to clarithromycin (MICs from 6 to >256 mg/l) except one strain for which again a mix of susceptible (MIC of 0.05 mg/l) and resistant strains was observed. For 3 (2.5%) strains, we obtained undetermined DHPLC profiles, i. e. neither a profile identical to a wild-type strain or a mutated strain. Different DHPLC retention profiles were observed for the two main mutations, A2146G (n = 3) and A2147G (n = 32). No strain harbouring the A2146C was observed. We also evaluated the minimal proportion of resistant strains with a mutated allele that can be detected. This was about 25% with DHPLC as with sequencing methods. Conclusion: DHPLC method is a reliable method to detect 23S rDNA mutations predictive of clarithromycin resistance.

S103 O482 Comparison of a molecular screening method with traditional culture for the detection of Salmonella spp. and Campylobacter jejuni in faeces R. de Boer, T. Schuurman, E. van Zanten, K. van Slochteren, H. Scheper, B. Dijk-Alberts, L. M¨oller, M. Kooistra-Smid (Groningen, NL) Objective: Salmonella spp. and Campylobacter jejuni are the major causes of bacterial gastro-enteritis in the Netherlands. Conventional diagnosis is based on detection of both species in faeces by traditional culturing which usually takes several days. We developed a sensitive molecular screening method (MSM) for the detection of both species which decreases the turn-around time significantly. This study describes the comparison of this real-time PCR based screening method with routine culture for the detection of Salmonella spp. and C. jejuni in faeces. Methods: A total number of 2,067 stool samples were analysed at our laboratory. Routine culture was performed on faecal samples and consisted of enrichment, selective culture and phenotypic identification. The molecular method consisted of a semi-automatic DNA extraction in combination with real-time PCR assays for Salmonella spp. and C. jejuni. PCR positive samples as well as samples which demonstrated PCR inhibition were cultured afterwards, consisting of the identical procedure as described for routine culture. Also, data regarding time to generate final results were collected for the MSM. Results: A total number of 2,055 samples were included for validation of Salmonella spp. The detection of Salmonella spp. improved by 15% with molecular screening; sensitivity was 100% and specificity 99%. For C. jejuni 2,009 samples were included and detection improved by 25%; sensitivity was 97% and specificity 75%. PCR inhibition was observed in less than 1.9% of all samples. The time to generate final results was less than 24 hours for all PCR negative samples (with exception of the inhibited samples), and in comparison to traditional culture there was no delay observed in generating results of culture confirmed PCR positive samples. Conclusions: 1. The MSM has a great potential for rapid detection of Salmonella spp. and C. jejuni in faeces. 2. Time to generate final results for negative samples was reduced dramatically to less than 24 hours. 3. The detection of Salmonella spp. and C. jejuni will improve considerably with molecular screening. 4. Automation of the extraction and detection procedures will further speed up the process and improve standardisation of the molecular screening procedure. O483 Staphylococci speciation and Panton-Valentine leukocidin detection by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry D. Dare, H. Li, H. Sutton, C. Stratton, Y. Tang (Manchester, UK; Nashville, US) Objectives: A rapid proteome analysis has proven useful for microbial identification. We assessed a matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry for the rapid identification and differentiation of staphylococcal species as well as the detection of Panton-Valentine Leukocidin (PVL) toxin from clinical staphylococcal isolates. Methods: Staphylococcal clinical isolates recovered from blood culture were included in the study. Phenotypic identification and differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were determined by colony morphology, catalase, and a Staphaurex latex agglutination. CoNS speciation was performed by using a bioM´erieux/VITEK API STAPH system. Methicillin resistance in S. aureus strains was confirmed by disk diffusion. The PVL gene was detected by a real-time TaqMan PCR assay. Results: Among a total of 115 staphylococcal isolates, 41 (35.7%), 44 (38.3%), and 30 (26.1%) were identified as CoNS, methicillin-