Susanne Jacobsson Date: 2014-03-12 Page 1 (8) Neisseria meningitidis 2013 Annual report concerning serogroup, genosubtype and antibiotic susceptibility for Swedish Neisseria meningitidis isolates and results with direct PCR for diagnosis of acute bacterial meningitis A total of 96 N. meningitidis (Nm) isolates from 89 patients or carriers were sent from the clinical diagnostic laboratories in Sweden, and examined in Örebro during 2013. Isolates from the cerebrospinal fluid (CSF)/blood/other sterile sites were characterised from a total of 67 patients. The total picture of invasive meningococcal disease in Sweden for the year 2013 is reached by amalgamating of this report with the compulsatory clinical notifications sent to the Public Health Agency of Sweden. An annual publication in collaboration with the national reference laboratory in Örebro is published from the Public Health Agency of Sweden, Solna, including case fatality rate, age distribution and more (http://www.folkhalsomyndigheten.se/amnesomraden/statistikoch-undersokningar/sjukdomsstatistik/meningokockinfektion-invasiv/). Isolates of Nm from CSF/blood, puncture, throat/respiratory tract including the conjunctiva and urogenital in relation to serogroup is presented in Table I. One isolate per patient is presented. Table II shows the genosubtype results for the 67 invasive Nm isolates (CSF/blood/puncture), Table III displays the MIC reults for the 67 invasive Nm isolates. For a more longitudinal presentation, see Table IV. Genetic subtyping i.e. genosubtypning, was performed by sequencing the three most variable regions (VR1, VR2 and VR3) of the porA gene. We are also using a co-agglutination assay with a monoclonal antibody (4BG4-E7), that reacts with a conserved part of most PorA proteins, in order to document the presence of expressed protein in the outer membrane. All 67 invasive Nm isolates were genosubtypable, whereas PorA expression was detected in 52/67=78%. Furthermore, multilocus sequencing typing (MLST) and fetA typing are conducted and reported to ECDC. The 2013 data were obtained from whole genome sequencing using Miseq (Illumina) and Bacterial Isolate Genome Sequence Database (BIGSdb) (http://pubmlst.org/software/database/bigsdb/). PCR diagnosis for CSF, blood and other sterile sites, has been performed with an optimised protocol for bacterial DNA, using the 16S rRNA gene, and species specific DNA, using the ctrA and crgA genes for meningococci (2, 3). POSTAL ADDRESS Department of Laboratory Medicine, Microbiology Örebro University Hospital SE-701 85 Örebro, Sweden e-mail [email protected] VISITING ADDRESS Södra Grev Rosengatan Entrance F2 Örebro, Sweden PHONE ORG.NR +46-(0)19-602 3541 18-232100-0164 FAX +46-(0)19-12 74 16 INTERNET www.orebroll.se/uso/mikrobiol Page 2 (8) Nm isolates from cerebrospinal fluid/blood/puncture (number of patients=number of isolates) The 67 invasive isolates characterised during 2013 comprised of: • • • • 17 NmB isolates (10 sulphonamide resistant, i.e. MIC >10 mg/L) belonged to 14 different genosubtypes. 13 NmC isolates (11 sulphonamide resistant) belonged to 5 different genosubtypes, of which P1.5,2,36-2 predominated (n=9). 34 NmY isolates (12 sulphonamide resistant) belonged to 4 different genosubtypes, of which P1.5-2,10-1,36-2 predominated (n=23) 3 NmW isolates (2 sulphonamide resistant) belonged to 2 different genosubtypes. Genosubtype distribution divided into serogroups is presented in Table II. Nm DNA in culture negative samples from normally sterile sites During 2013, 59 culture negative CSF and other samples were examined concerning meningococci, with PCR technique for bacterial DNA (2). Meningococcal DNA, the ctrA and crgA genes, was documented in 6 of the samples (four CSF, two DNA preparations). The genogroup and genosubtype was diagnosed directly from the sample in 4 of these cases, whereas two samples were not possible to genotypable type genetically (1). Antibiotic susceptibility All the Nm isolates were tested regarding antimicrobial susceptibility using Etest (Biomerieux) on Mueller-Hinton (BD) agar with blood (MH-F). MIC were obtained for benzylpenicillin (PcG), cefotaxime, chloramphenicol, ciprofloxacin, rifampicin and meropenem. As an additional epidemiological marker susceptibility to sulphonamides (sulfametoxazole) was also tested using Etest (Biomerieux). During 2013 the proportion of invasive Nm with reduced susceptibility to PcG (MIC >0.064 mg/L) was 18/67=27%. See also Figure 1, where the number of invasive Nm isolates with reduced susceptibility for PcG from 1999 to 2013 are presented. Part of the increase of invasive Nm with reduced susceptibility to PcG in 2013 might be explained by the ~20 NmY isolates, both invasive and non-invasive, that grew poorly on our standard media for susceptibility testing (MH-F). In those cases we used a richer medium which we have seen could result in slightly higher MIC. Exclusively looking on the invasive isolates grewing well 14/58=24% displayed reduced susceptibility to PcG, which still represents an increase during 2013. No β-lactamase producing Nm has so far been isolated in Sweden. Page 3 (8) 40 35 30 25 20 15 10 5 0 1999 2001 2003 2005 2007 2009 2011 2013 Figure 1. The proportion (%) of invasive Nm with reduced sensitivity (MIC >0.064 mg/L) to pcG during 1999 to 2013. In conclusion The Y:P1.5-2,10-1,36-2 (n=23) together with C:P1.5,2,36-2 (n=9) and Y:P1.5-1,2-2,36-2 (n=8) were predominant among meningococci causing invasive disease in Sweden during 2013. Compared to previous years (Table IV) the increase of the total number of meningococci seen from 2010 has stalled during 2013. Nm serogroup B (n=17) remains, similarily to the latest years, at a relatively low level. Since 2010 the mean number of invasive NmB isolates from CSF/blood have been 15, range 11-19 as compared to 22 per year, range 14-33 during 2000-2009 and 40, range 25-66 during 1990-1999. Nm serogroup C was isolated in 13 invasive cases, which is within the lower range recorded since 1999 (10-22 isolates/year) and represent a decrease from 2012 (n=22). The increased incidence of invasive Nm serogroup Y observed since 2009 appears to have stabilized during 2013. However, the proportion of NmY to the total nummer of invasive Nm isolates in Sweden was in congruence with previous years and the NmY represented 51% of the cases 2013, compared to 49% in 2012 and 51% in 2011. As previously shown the emerging increase of serogroup Y in Sweden is due to a specific strain type, i.e. Y:P1.5-2,10-1,36-2:F4-1:ST23(cc23) (8). This genosubtype P1.5-2,10-1,36-2 were in 2013 the most prevalent genosubtype among all the invasive Nm isolates and represented 23/34 of the Nm serogroup Y isolates. The second most prevalent genosubtype of NmY, P1.5-1,2-2,36-2, represented 24% (8/34) of the NmY isolates and has been the second most prevalent genosubtype also previous years (25% 2012, 10% 2011). No clusters or coupled cases were reported in Sweden during 2013. However, one case in the south of Sweden was linked to a case in a younger relative in Denmark. Finally, it is of importance to follow (phenotypically and genetically) the changes within the meningococcal population circulating in the community, for example, in regards to future vaccine implementations and possible emergence of resistance to antimicrobials used in the prevention or treatment of invasive meningococcal disease. Page 4 (8) Selected publications 1. Mölling P, Jacobsson S, Bäckman A, Olcén P. Direct and rapid identification and genogrouping of meningococci and porA amplication by LightCycler PCR. J Clin Microbiol 40:4531-4535, 2002. 2. Thulin Hedberg S, Olcén P, Fredlund H, Mölling P. Real-time PCR detection of five prevalent bacteria causing acute meningitis. APMIS 117:856-860, 2009. 3. Cavrini F, Liguori G, Andreoli A, Sambri V. Multiple nucleotide substitutions in the Neisseria meningitidis serogroup C ctrA gene cause false-negative detection by real-time PCR. J Clin Microbiol 48:3016-3018, 2010. 4. Susanne Jacobsson. Doctoral dissertation. Characterization of Neisseria meningitidis from a virulence and immunogenic perspective that includes variations in novel vaccine antigens. Örebro Studies in Medicine 31, Örebro 2009. 5. Sara Thulin Hedberg. Doctoral dissertation. Antibiotic susceptibility and resistance in Neisseria meningitidis – phenotypic and genotypic characteristics. Örebro Studies in Medicine 38, Örebro 2009. 6. Jacobsson S, Wedege E. A review of vaccines against group B meningococcal disease. European Infectious Disease 4:50-53, 2010. 7. Thulin Hedberg S, Olcén P, Fredlund H, Unemo M. Antibiotic susceptibility of invasive Neisseria meningitidis isolates from 1995 to 2008 in Sweden – the meningococcal population remains susceptible. Scand J Infect Dis 42:61-64, 2010. 8. Thulin Hedberg S, Törös B, Fredlund H, Olcén P, Mölling P. Genetic characterisation of the emerging invasive Neisseria meningitidis serogroup Y in Sweden 2000 to 2011. Euro Surveill 16:1-7, 2011. 9. Bröker M, Jacobsson S, DeTora L, Pace D, Taha MK. Increase of meningococcal serogroup Y cases in Europe: A reason for concern? Hum Vaccin Immunother 8:685-8, 2012. 10. Bröker M, Jacobsson S, Kuusi M, Pace D, Simões MJ, Skoczynska A, Taha MK, Toropainen M and Tzanakaki G. Meningococcal serogroup Y emergence in Europe: Update 2011. Hum Vaccin Immunother 8:1907-11, 2012. 11. Kelly A, Jacobsson S, Hussain S, Olcén P, Mölling P. Gene variability and degree of expression of vaccine candidate factor H binding protein in clinical isolates of Neisseria meningitidis. APMIS 121:56-63, 2013. 12. Törös B, Hedberg ST, Jacobsson S, Fredlund H, Olcén P, Mölling P. Evaluation of molecular typing methods for identification of outbreak-associated Neisseria meningitidis isolates. APMIS 121:503-10, 2013. 13. Bröker M, Bukovski S, Culic D, Jacobsson S, Koliou M, Simões MJ, Skoczynska A, Toropainen M, Taha MK, Tzanakaki G. Meningococcal serogroup Y emergence in Europe: High importance in some European regions in 2012. Hum Vaccin Immunother 10 [Epub ahead of print] 2014. Report sent to: Public Health Agency of Sweden, att: Johan Carlsson, Anders Tegnell, Karin Tegmark Wisell, Tiia Lepp and its counterpart in the Nordic countries National Board of Health and Welfare, att: Agneta Holmström, Axana Haggar ECDC, att: Marc Sprenger, Johan Giesecke, Karl Ekdahl, Lucia Pastore Celentano, Denis Coulombier, Marc Struelens EMGM – The European Meningococcal Disease Society, with the Ref. laboratories in Europe, att: Georgina Tzanakaki WHO Euro, att: Zsuzsanna Jakab Örebro 2014-03-12 Susanne Jacobsson, Hans Fredlund, Paula Mölling, Sara Thulin Hedberg, Per Olcén, Bianca Törös, Martin Sundqvist, Magnus Unemo Page 5 (8) Table I. Serogroup distribution for N. meningitidis isolates from 89 patients or carriers sent to the National Reference Laboratory for characterisation during 2013. One isolate per patient is presented. Serogroup CSF/blood Puncture Pharynx/airways incl. conjunctiva Total B 17 - 6 23 C 13 - 1 Y 33 1 11 W 3 - - Non-groupable (ng) - - 4 Total 66 1 22 a) 1 conjunctiva b) 1 sputum, 1 cornea c) 1 sputum a) b) c) 14 45 3 4 89 Page 6 (8) Table II. Genosubtype pattern for all the invasive N. meningitidis isolates (n=67) sent to the National Reference Laboratory for characterisation during 2013. Displaying the three variable regions VR1, VR2 and VR3 of the porA gene. Genosubtype P1.5-2,10-1,36-2 NmY NmW 9 1 8 11 8 2 2 Total 23 1 P1.18-1,3,38 P1.5-1,10-1,36-2 NmC 23 P1.5,2,36-2 P1.5-1,2-2,36-2 NmB 2 4 1 3 P1.7-2,4,37 2 2 P1.22,14-6,36-2 2 2 P1.5-1,10-8,36-2 1 1 P1.7,16,35 1 1 P1.7,16-26,35 1 1 P1.7,16-29,35 1 1 P1.7,16-32,35 1 1 P1.7-2,13-2,35-1 1 1 P1.7-2,16-29,35 1 P1.12-1,13-15,35-1 1 1 P1.17,16-4,36 1 1 1 P1.19,15,36 1 1 P1.19,15-1,36 1 1 P1.19-1,26,36-2 1 1 P1.21,16,37-1 1 1 P1.22,13-22,35-1 1 1 Total 34 17 13 3 67 Page 7 (8) Tabell III. MIC range for all the invasive N. meningitidis isolates (n=67) sent to the National Reference Laboratory for characterisation during 2013 and clinical breakpoints within the SIR-system settled by the European Committee on Antimicrobial Susceptibility Testing/Nordic Committee on Antimicrobial Susceptibility Testing. a) Antibiotic MIC range (mg/L) S≤ ≤/R> Penicillin G 0.012 – 0.25 0.06/0.25 Cefotaxime <0.002 – 0.023 0.12/0.12 Chloramphenicol 0.125 – 1.5 2/4 Ciprofloxacin 0.002 – 0.008 0.03/0.06 Rifampicin 0.002 – 0.125 0.25/0.25 Meropenem <0.002 – 0.032 0.25/0.25 a) S – susceptible, R – resistant. EUCAST/NordicAST breakpoints for Nm Susanne Jacobsson Date 2014-03-12 Page: 8 (8) Table III. Distribution of all N. meningitidis isolates sent for characterisation to the Reference Laboratory in Sweden from 1999 to 2013. Only one isolate per patient is included. 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 Total no. of Nm-isolates 87 102 116 83 74 73 70 75 81 76 79 94 99 130 89 Nm from CSF/blood 46 44 58 36 41 47 48 44 43 38 45 56 61 85 66 Nm from respiratory tract 39 56 55 43 27 24 19 28 34 35 24 35 33 35 22 Nm from urogenital tract 2 1 2 3 4 - 1 3 3 2 9 2 5 5 - Other invasive Nm - 1 1 1 2 2 2 - 1 1 1 1 - 5 1 A - - - - - - - - - - - 1 - - - B 25 23 33 20 27 24 25 21 16 14 16 11 14 19 17 C 12 15 14 10 11 11 15 15 15 16 10 19 15 22 13 Y 7 1 8 4 2 6 4 5 9 7 16 22 31 39 33 W 2 5 2 1 1 5 1 2 2 1 2 2 1 5 3 other - - 1 - - - 1 - - - - 1 - - - ng - - - 1 - 1 2 1 1 - 1 - - - - Nm från CSF/blood serogroup