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(microsoft word - \305rsrapportmc2011 annual report 120307.doc)

Susanne Jacobsson Date: 2012-03-07 Page 1 (9)
Neisseria meningitidis 2011
Annual report concerning
serogroup, genosubtype and antibiotic susceptibility for
Swedish Neisseria meningitidis isolates and results with
direct PCR for diagnosis of acute bacterial meningitis.

Report sent to:
Department of epidemiology, SMI, att: Annika Linde, Tiia Lepp, Birgitta Henriques Normark
ECDC, att: Marc Sprenger, Johan Giesecke, Karl Ekdahl, Lucia Pastore Celentano, Andrea Amato Gauci, Ida Czumbel
All members of EMGM – The European Meningococcal Disease Society
National epidemic units in the EU countries
WHO HQ, WHO Euro, and CDC.

A total of 111 N. meningitidis (Nm) isolates from 99 patients were sent from the clinical diagnos-
tic laboratories in Sweden, and examined in Örebro during 2011. Isolates from the cerebrospinal
fluid (CSF), and/or blood were characterised from a total of 61 patients. The total picture of inva-
sive meningococcal disease in Sweden for the year 2011 is reached by amalgamating this report
with the compulsatory clinical notifications sent to the National Swedish Institute for Infectious
Disease Control (SMI). An annual publication of this total picture, including case fatality rate, age
distribution and more is published from SMI, Solna in collaboration with the national reference
laboratory in Örebro.
Isolates of Nm from CSF/blood, throat/respiratory tract including the conjunctiva and urogenital
in relation to serogroup is presented in Table I. One isolate per patient is presented.
Table IIa, b and c shows the genosuptype results for the 61 invasive Nm isolates (CSF/blood).
For a more longitudinal presentation, see Table III.
Genetic subtyping i.e. genosubtypning were 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 react with
a conserved part of most PorA proteins, in order to document the presence of the protein in the
outer membrane. Among the 61 invasive Nm isolates we identified one isolate containing a dele-
tion of VR1 and three isolates, coupled to one outbreak, with a deletion of VR2. The other Nm
isolates were genosubtypable and PorA expression was detected in 57/61=93%.
PCR diagnosis for CSF, blood and puncture material, has been performed with an optimised
protocol (ref. 9) for bacterial DNA (the gene for 16S rRNA) and species specific DNA (the ctrA
and crgA genes for meningococci).
Department of Laboratory Medicine, Microbiology Södra Grev Rosengatan +46-(0)19-602 1134 e-mail susanne.jacobsson@orebrol .se
INTERNET www.orebrol .se/uso/mikrobiol
Page 2 (9)
Nm isolates from cerebrospinal fluid/blood (number of patients=number
of isolates).

Serogroup B.
The 14 invasive NmB isolates (10 sulphonamide resistant, i.e. MIC >10 mg/L) belonged to 8
different genosubtypes, presented in Table IIa.
Serogroup C.
The 15 invasive NmC isolates (14 sulphonamide resistant) belonged to 5 different genosubtypes,
wherein P1.5,2,36-2 dominated (n=10), see Table IIb.
Serogroup Y.
The 31 invasive NmY isolates (14 sulphonamide resistant) belonged to 3 different genosubtypes,
wherein P1.5-2,10-1,36-2 dominated (n=27), see Table IIc.
MC DNA in culture negative samples from normal sterile sites.

During 2011, 61 culture negative CSF and other samples were assayed concerning meningococci,
with PCR technique for bacterial DNA (ref. 9). Meningococcal DNA (the ctrA and crgA genes)
was documented in 9 of the samples (five CSF, three DNA preparation, one blood). In 6 out of 9
samples genogroup and genosubtype of Nm was diagnosed directly from the sample (ref. 1).
Sequencing of bacterial DNA (after general amplification of 16S rRNA gene) has also been used
for species designation.

Antibiotic susceptibility.
All the Nm isolates were tested with the Etest concerning MIC for pcG, cefotaxime, chloram-
phenicol, ciprofloxacin, meropenem and rifampicin. In addition sensitivity for sulphonamide was
tested. This is a good epidemiological marker in addition to the other ways of characterising Nm,
see above.
The proportion 2011 of invasive Nm with laboratory reduced susceptibility for pcG (MIC
>0.064 mg/L) was 7/61=11%
. See also Figure 1, where the number of invasive Nm isolates with
laboratory reduced susceptibility for pcG since 1999 to 2011 are shown.
All isolates had cefotaxime MIC <0.008 except one isolate with MIC 0.012. All isolates had
ciprofloxacin MIC <0.006. Meropenem MIC varied between 0.002 and 0.032, chloramphenicol
MIC between 0.19 and 1.5 and rifampicin MIC between 0.002 and 0.064 mg/L.
No betalactamase producing Nm has so far been isolated in Sweden.
The breakpoints for Nm within the SIR system (S≤/R>) for communication with the physicians
responsible for the treatment have been settled by the European Committee on Antimicrobial Sus-ceptibility Testing/National Reference Group for Antibiotics in Sweden. The breakpoints are at present pcG 0.06/0.25, cefotaxime 0.12/0.12, ciprofloxacin 0.03/0.06, meropenem 0.25/0.25, chloramphenicol 2/4 and rifampicin 0.25/0.25. Page 3 (9)
Figure 1. The proportion (%) of invasive Nm with laboratory reduced sensitivity
(MIC >0.064 mg/L) for pcG during 1999 to 2011. In conclusion.

From the Swedish Reference Laboratory perspective were Y:P1.5-2,10-1,36-2 (n=27) and
C:P1.5,2,36-2 (n=10) dominant among meningococci causing invasive disease in Sweden during
2011. The second most prevalent isolates were, B:P1.7,16,35 (n=3), B:P1.7-20,-,37 (n=3) and
Y:P1.5-1,2-2,36-2 (n=3).

Compared to previous years (Table III) the total number of meningococci has increased somewhat
during 2011.
Nm serogroup B (n=14), as has also been noted the last years, are at a low level compared with the
past. The mean number of NmB isolates from CSF/blood were 14, range 11-16 during the years
2007-2011 compared to 30 CSF/blood MCB isolates per year, range 20-54 during 1995-2006.
Nm serogroup C was isolated in 15 cases, which is within the lower range recorded since 1995
(9-25 isolates/year) and represent a decrease from 2010 (n=19).
Nm serogroup Y continues, like the last two years, to increase and is now representing 51% of the
invasive Nm isolates in Sweden during 2011. A study has been conducted which showed that this
emerging increase of serogroup Y mainly is due to the increase of one specific clone in Sweden
i.e. Y:P1.5-2,10-1,36-2:F4-1:ST23(cc23) (ref. 15).
Finally, it is of importance to follow (phenotypically and genetically) what is happening with the
meningococci circulating in the community, for example in regards to future vaccine
implementations and antibiotic susceptibility.
Page 4 (9)

Selected publications.
1. Möl ing 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.
Molecular genetic methods in diagnosis and direct characterization of acute bacterial central nervous system infections.
APMIS 112:753-770, 2004.
Interlaboratory comparison of PCR-based identification and genogrouping of Neisseria meningitidis.
J Clin Microbiol 43:144-149, 2005.
4. Thulin S, Olcén P, Fredlund H, Unemo M. Total variation in the penA gene of Neisseria meningitidis: correlation between susceptibility to β-lactam antibiotics and
penA gene heterogeneity.
Antimicrob Agents Chemother 50:3317-3324, 2006.
5. Thulin S, Olcén P, Fredlund H, Unemo M. Combined real-time PCR and pyrosequencing strategy for objective, sensitive, specific, and high throughput identification of reduced sensitivity to penicil ins in Neisseria meningitidis. Antimicrob Agents Chemother 52:753-756, 2008.
6. Jacobsson S, Olcén P, Löfdahl M, Fredlund H, Möl ing P. Characteristics of Neisseria meningitidis isolates causing fatal disease. Scand J Infect Dis 40:734-744, 2008.
7. Wu HM, Harcourt BH, Hatcher CP et al. Emergence of ciprofloxacin-resistant Neisseria meningitidis in North America.
NEJM 360:886-892, 2009.
8. Jacobsson S, Thulin Hedberg S, Möl ing P, Unemo M, Comanducci M, Rappuoli R, Olcén P. Prevalence and sequence variations of the genes encoding the five antigens included in the novel 5CVMB vaccine covering
group B meningococcal disease.
Vaccine 27:1579-1584, 2009.
9. Thulin Hedberg S, Olcén P, Fredlund H, Möl ing P. Real-time PCR detection of five prevalent bacteria causing acute meningitis.
APMIS 117:856-860, 2009.
Doctoral dissertation. Characterization of Neisseria meningitidis from a virulence and immunogenic perspective that in-cludes variations in novel vaccine antigens. Örebro Studies in Medicine 31, Örebro 2009. Doctoral dissertation. Antibiotic susceptibility and resistance in Neisseria meningitidis – phenotypic and genotypic character-istics. Örebro Studies in Medicine 38, Örebro 2009. A review of vaccines against group B meningococcal disease.
European Infectious Disease 4:50-53, 2010.
13. Thulin Hedberg S, Olcén P, Fredlund H, Unemo M. Antibiotic susceptibility of invasive Neisseria meningitidis isolates from 1995 to 2008 in Sweden – the meningococcal popu-
lation remains susceptible.
Scand J Infect Dis 42:61-64, 2010.
14. 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.
15. Thulin Hedberg S, Törös B, Fredlund H, Olcén P, Möl ing P. Cavrini F, Liguori G, Andreoli A, Sambri V. Genetic characterisation of the emerging invasive Neisseria meningitidis serogroup Y in Sweden 2000 to 2011. Euro Surveil 16:1-7, 2011.

Örebro 2012-03-07
Susanne Jacobsson, Per Olcén, Hans Fredlund, Paula Mölling, Sara Thulin Hedberg, Magnus
Unemo, Bianca Törös.
Page 5 (9)

Table I. Serogroup distribution for N. meningitidis isolates from 99 patients sent to the
National Reference Laboratory for characterisation during 2011. One isolate per patient is presented. Serogroup
incl. conjunctiva
a) 1 bronchal, b) 1 conjunctiva, c) 1 sputum, d) 1 sputum. Page 6 (9)

Table IIa. Genosubtype pattern for al the invasive N. meningitidis serogroup B isolates
(n=14) presented as the designed variation in the three regions VR1, VR2
and VR3 of the porA gene.
The group B-isolates had genosubtype P1.7,16,35
Page 7 (9)

Table IIb. Genosubtype pattern for al the invasive N. meningitidis serogroup C isolates
(n=15) presented as the designed variation in the three regions VR1, VR2
and VR3 of the porA gene.

The group C-isolates had genosubtype P1.5,2,36-2
Page 8 (9)

Table IIc. Genosubtype pattern for al the invasive N. meningitidis serogroup Y isolates
(n=31) presented as the designed variation in the three regions VR1, VR2
and VR3 of the porA gene.

The group Y-isolates had genosubtype P1.5-2,10-1,36-2
Invasive isolates of other serogroups
Group W-135
Susanne Jacobsson Date: 2012-03-07 Page: 9 (9)

Table III.
Distribution of al N. meningitidis isolates sent to the Reference Laboratory in Sweden from 1995 to 2011. Only one isolate per
patient is included.
1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011
Total no. of Nm-
Nm from CSF/
Nm from respiratory
Nm from urogenital
Other invasive Nm
Nm från CSF/blood


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