BACTERIAL MENINGITIS IN THE NETHERLANDS

Transcription

1 NETHERLANDS REFERENCE LABORATORY FOR BACTERIAL MENINGITIS BACTERIAL MENINGITIS IN THE NETHERLANDS ANNUAL REPORT 2007 AMC Academic Medical Center University of Amsterdam RIVM National Institute of Public Health and Environmental Protection

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3 NETHERLANDS REFERENCE LABORATORY FOR BACTERIAL MENINGITIS Academic Medical Center (AMC) and National Institute of Public Health and the Environment (RIVM), Department of Medical Microbiology PO Box 22660, 1100 DD Amsterdam The Netherlands Telephone / Fax reflab@amc.uva.nl The contents of this report may be quoted, provided the source be given: Netherlands Reference Laboratory for Bacterial Meningitis (AMC/RIVM) Bacterial meningitis in the Netherlands; annual report 2007 Amsterdam: University of Amsterdam,

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5 CONTENTS CONTENTS INTRODUCTION ISOLATES, CSF SPECIMENS AND SERA RECEIVED BACTERIAL MENINGITIS - general data NEISSERIA MENINGITIDIS General features Antibiotic susceptibility Serogroups Serogroup and age Group B meningococci Distribution of PorA genosubtypes among serogroup B and C meningococci Distribution of FetA genosubtypes among serogroup B and C meningococci 28 5 HAEMOPHILUS INFLUENZAE STREPTOCOCCUS PNEUMONIAE General features Antibiotic susceptibility Distribution according to serotype 36 7 ESCHERICHIA COLI STREPTOCOCCUS AGALACTIAE (group B) LISTERIA MONOCYTOGENES STREPTOCOCCUS PYOGENES ANTIGEN AND DNA DETECTION IN CSF AND SERUM VACCINATION PROSPECTS N. meningitidis H. influenzae S. pneumoniae PUBLICATIONS ACKNOWLEDGEMENTS

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8 1 INTRODUCTION This is the 36 th Annual Report of the Netherlands Reference Laboratory for Bacterial Meningitis of the Academic Medical Center (AMC) and the National Institute of Public Health and the Environment (RIVM). The Reference Laboratory is located within the Department of Medical Microbiology of the Academic Medical Center (AMC) in Amsterdam. Nearly all clinical microbiology laboratories of the Netherlands collaborate by sending bacterial isolates and/or cerebrospinal fluid samples from patients with meningitis and we are most grateful to our colleagues for their cooperation. The Reference Laboratory started collecting isolates of Neisseria meningitidis in 1959 and of other bacteria causing meningitis in In the archives of the Reference Laboratory data from approximately 43,000 cases and more than 58,500 isolates are now available for studies on the epidemiology of bacterial meningitis and on the pathogenicity and antibiotic susceptibility of isolates. The objectives of the Reference Laboratory are: - to perform surveillance of bacterial meningitis; - to describe the epidemiology of bacterial meningitis in the Netherlands; - to provide keys for the development of potential vaccine components; - to provide data about antibiotic susceptibility of isolates. The information is presented in tables and figures and shortly discussed in the text. We would appreciate receiving your opinion and suggestions on this report. Amsterdam, August, 2008 dr. A. van der Ende, biochemist dr. L. Spanjaard, medical microbiologist 7

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10 2 ISOLATES, CSF SPECIMENS AND SERA RECEIVED The Netherlands Reference Laboratory for Bacterial Meningitis collects isolates from cerebrospinal fluid (CSF) and blood from patients with proven meningitis (CSF and possibly blood culture positive) or with bacteraemia and suspected meningitis (blood culture positive only). Unless otherwise indicated, every isolate from CSF, from CSF and blood, and from blood represents a patient with meningitis, a patient with meningitis and bacteraemia, and a bacteraemia patient, respectively. Incidences have been calculated by dividing the number of isolates collected over one year (in a certain patient s age group) by the number of inhabitants over one year (in that age group) multiplied by 100,000. Population figures were obtained from Statistics Netherlands (Centraal Bureau voor de Statistiek, using StatLine. By estimation, the Reference Laboratory receives about 85% of the isolates of Dutch meningitis patients, hence incidences presented in this report are likely to be underestimated. In 2007, the Reference Laboratory received isolates from CSF and / or blood from 1507 patients, and 225 specimens of CSF and serum (table 2.1). Of these patients, 499 were confirmed cases of bacterial meningitis. Table 2.1 Number of specimens Isolate (CSF and/or blood) 1507 CSF (without isolate) 196 Sera (and other fluid) 29 Total

11 In 2007, 62 clinical microbiology laboratories submitted isolates to the Reference Laboratory. Table 2.2 shows the 1518 isolates according to species and to laboratory where cases were diagnosed. Table 2.2 Number of isolates # from CSF and/or blood received in 2007, according to laboratory Location Laboratory Nm Hi Sp Ec Sag Lm Spy Sau Cns Cn ot nv Total Alkmaar MCA lab. Med. Microbiologie Amersfoort Meander Medisch Centrum Amsterdam AMC Acad. Medisch Centrum Academisch Ziekenhuis VU GG & GD Onze Lieve Vrouwe Gasthuis Slotervaart Ziekenhuis St. Lucas Ziekenhuis Apeldoorn Medische Laboratoria ZCA Arnhem Alysis Zorggroep, lok Rijnstate Bergen op Zoom Ziekenhuis Lievensberg Breda Amphia Ziekenhuis Molengracht Capelle ad IJssel IJsselland Ziekenhuis Delft SSDZ (Reinier de Graaf groep) Den Bosch Regionaal laboratorium Den Bosch Den Haag MC.Haaglanden loc. Westeinde Bronovo Ziekenhuis Leijenburg Ziekenhuis Deventer Med.Microbiologie Geertruiden Ziekenhs Doetinchem Slingeland Ziekenhuis Dordrecht Reg. Lab. Med. Microbiologie Ede Gelderse Vallei Enschede Lab. Med. Microbiologie Goes Oosterschelde ZKH, Med. Microbiologie Gouda Groene Hart Ziekenhuis Groningen AZG, Gebouw MVC Lab. v. Infectieziekten Haarlem Streeklab. VG Kennemerland Hardenberg Streeklab. Groningen dep. Ropkezweers Harderwijk St. Jansdal Ziekenhuis Heerlen Streeklab. VG Heerlen Hilversum Centraal Bact. Ser. Lab Hoorn Westfries gasthuis Leeuwarden Streeklab. VG Friesland Leiden LUMC, KML, Lab.Bact., E Diakonessen Ziekenhuis Leiderdorp Rijnland Ziekenhuis Maastricht Acad. Ziekenhuis Maastricht Meppel Str.lab. Groningen en Drenthe Nieuwegein St. Antonius Ziekenhuis Nijmegen Canisius Wilhelmina Ziekenhuis UMC St. Radboud Roermond St. Laurentius Ziekenhuis Roosendaal St. Fransiscus Ziekenhuis

12 Location Laboratory Nm Hi Sp Ec Sag Lm Spy Sau Cns Cn ot nv Total Rotterdam Erasmus MC Med. Microbiologie Ikazia Ziekenhuis Med. Microbiologie M.C. Rijnmond Zuid loc. St.Clara St.Franciscus Gasthuis Schiedam Schieland Ziekenhuis Sittard Maasland Ziekenhuis Spijkenisse Ruwaard van Putten Ziekenhuis Tilburg Streeklab. Tilburg Utrecht Diakonessenhuis UMC Med. Microbiologie G Veldhoven Lab. Med. Microbiologie Venlo Vie Curie medisch centrum Weert St. Jans gasthuis Woerden Zuwe Hofpoort Ziekenhuis Zaandam Zaans Medisch Centrum De Heel Zwolle Lab. v. Med. Microbiologie Absolute total* Real total # Nm: N. meningitidis; Hi: H. influenzae; Sp: S. pneumoniae; Ec: E. coli; Sag: S. agalactiae; Lm: L. monocytogenes; Spy: S.pyogenes; Sau: S. aureus; Cns: Coagulase negative staphylococcus; Cn: C. neoformans; ot: other bacteria; nv: non-viable * Three meningococcal, one Haemophilus, four pneumococcal, two S. agalactiae and one C. neoformans isolates were sent by two different laboratories The distribution of the isolates received in the 5 year period 2003 through 2007 is presented in table 2.3. The number of total isolates decreased from 2419 in 2003 to 1507 in The number of meningococcal disease cases reduced from 347 cases in 2003 to only 171 cases in The number of Streptococcus pneumoniae isolates from CSF has remained nearly constant over the years. In 2005, the total number of pneumococcal isolates was lower than in 2003 and 2004, because from 2003 on the Reference Laboratory requested pneumococcal blood isolates from a selected number of laboratories only. The number of Listeria monocytogenes was higher in the years 2005 and 2006, most likely due to a intensified surveillance performed by the RIVM. In 2007, the number of L. monocytogenes isolates were equal to that in

13 Table 2.3 Number of isolates from CSF and/or blood received in the years Isolate CSF Blood Total CSF Blood Total CSF Blood Total CSF Blood Total CSF Blood Total N. meningitidis H. influenzae S. pneumoniae E. coli S. agalactiae L. monocytogenes S. pyogenes S. aureus Coag.neg.Staph C. neoformans others non viable Total CSF: CSF or CSF and blood blood: blood only YEAR The incidence of isolation of the different bacterial species from CSF and/or blood over the years 2003 to 2007 is shown in table 2.4. The incidence of N. meningitidis infection was 51% lower than in This is partly due to a decline in the number of cases of N. meningitidis group C. In 2002, nationwide vaccination against serogroup C meningococcal disease was started. The incidence of Haemophilus influenzae infection was 67% lower than in the years before vaccination was introduced (2.1 in 1992; 0.70 in 2007), although the incidence of H.influenzae infection is gradually increasing since 1999, mainly due to an increase in the number of cases of H. influenzae septicemia. Table 2.4 Number of isolates from CSF and/or blood per 100,000 inhabitants, YEAR Species N. meningitidis H. influenzae S. pneumoniae E. coli S. agalactiae L. monocytogenes S. pyogenes S. aureus Coag. neg. Staph C. neoformans others non viable Total Table 2.5 shows the distribution of isolates according to the specimen from which they were cultured. The predominant species were N. meningitidis and S. pneumoniae. 12

14 Table 2.5 Total number of isolates from CSF and/or blood received in 2007, according to bacterial species and specimen source Species CSF or CSF and blood Blood only Total % Neisseria meningitidis Haemophilus influenzae Streptococcus pneumoniae Escherichia coli Streptococcus agalactiae Listeria monocytogenes Streptococcus pyogenes Staphylococcus aureus Coagulase-negative staphylococcus 9 4,5, Cryptococcus neoformans Others total Others Pseudomonas aeruginosa 1-1 Proteus mirabilis 2-2 Citrobacter freundii 2 7, 8-2 Klebsiella pneumoniae 3-3 Enterobacter cloacae 1-1 Serratia marcescens 1-1 Clostridium cadaveris 1-1 Neisseria sicca Neisseria subflava Moraxella osloensis Haemophilus parainfluenzae Streptococcus cristatus 1-1 Streptococcus haemolyticus group C 1-1 Streptococcus mitis 1-1 Streptococcus oralis Streptococcus parasanguinis Streptococcus salivarius Streptococcus suis 1-1 Enterococcus faecalis 3-3 Enterococcus species 1-1 Aerococcus urinae Leuconostoc species Lactococcus lactis ssp cremoris Candida albicans 1-1 Nocardia farcinica 1-1 Contamination Non viable Total % In one patient N. meningitidis and Moraxella osloensis were isolated from blood. 2 In one patient S. pneumoniae and H. influenzae were isolated from CSF and in another patient they were both isolated from blood. 3 In one patient S. pneumoniae and a Leuconostoc were isolated from blood. 4 In one patient S. epidermidis and Klebsiella pneumoniae were isolated from CSF. 5 In one patient S. xylosus and E. faecalis were isolated from the CSF. 6 Coag. Neg. staphylococcus includes four S. epidermidis, three S. capitis and two S.xylosus 7 In one patient C. freundii, K. oxytoca and Stenotrophomonas maltophilia were isolated from the CSF 8 In one patient C. freundii and B. thetaiotaomicron and were isolated from CSF. 9 In one patient S. oralis and S. salivarius were isolated from CSF. 13

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16 3 BACTERIAL MENINGITIS - general data Figure 3.1 shows the distribution of the isolates from the CSF of the 466 patients with confirmed meningitis in The proportion of N. meningitidis isolates decreased further to only 19% and was the lowest since 1994 (45%) (2002: 42%; 2003: 33%; 2004: 25%; 2005: 22%; 2006: 20%). The year 2003 was the first year, since two decades, in which N. meningitidis was not the main cause of bacterial meningitis in the Netherlands. Twenty-nine culture negative CSF samples and four serum/other samples appeared to be positive by antigen detection or PCR (Table 11.1). Of these, 15 were positive for S. pneumoniae, 17 for N. meningitidis and 1 for C. neoformans. Including these cases, the proportion pneumococcal and meningococcal meningitis among 499 patients with confirmed meningitis is 52% and 21%, respectively. S.pneumoniae 52% S.agalactiae 6% E.coli 3% L.monocytogenes 3% S.aureus 1% S.pyogenes 1% Coag. Neg. Staph. 2% H.influenzae 4% Non viable 1% N.meningitidis 19% C.neoformans 2% other 6% Figure 3.1 Proportional distribution of CSF isolates, 2007 Figure 3.2 shows the annual total number of bacterial isolates from CSF during the period The 10 years trend line indicates a slight decrease over the last decade, as it did over the previous years. The incidence per 100,000 inhabitants also shows a decreasing trend and varied between 4.9 and 2.8 during the period (Figure 3.2). Data concerning N. meningitidis, H. influenzae and S. pneumoniae during the same period are presented in figure 3.3. Since the introduction of vaccination against H. influenzae type b in 1993, the incidence of Haemophilus meningitis decreased to 0.2 per 100,000 and remained at this low level. The number of cases of meningococcal meningitis decreased from 390 cases (incidence of 2.5) in 2001 to 89 cases (incidence of 0.5) in 2007, partly due to a decline in the number of cases of serogroup B meningitis and partly due to a decline in the number of cases of serogroup C meningitis. Nationwide vaccination against serogroup C meningococci was started 15

17 in Pneumococcal meningitis is slowly increasing since 1991 as the annual incidence rose from 1.0 to 1.6 per 100,000 inhabitants in 2004 and was 1.5 in Number of isolates 1000 Number of isolates from CSF or CSF and blood Numer of isolates per 100,000 inhabitants Per 100,000 inhabitants Years Figure 3.2 Isolates from CSF, Number of isolates Per 100,000 inhabitants 3,5 3 2,5 2 1,5 1 0, Years Number of N. meningitidis isolates Number of H. influenzae isolates Number of S. pneumoniae isolates Number of N. meningitidis per 100,000 inhabitants Number of H. influenzae per 100,000 inhabitants Number of S. pneumoniae per 100,000 inhabitants Figure 3.3 Meningococcal, Haemophilus and pneumococcal meningitis,

18 Table 3.1 shows the frequency of isolation of the different bacterial species from CSF by annual quarter. As in previous years, most strains were received during the first quarter of the year. Table 3.1 Isolates from CSF by annual quarter, 2007 ANNUAL QUARTER SPECIES first second third fourth Total % N. meningitidis H. influenzae S. pneumoniae E. coli S. agalactiae L. monocytogenes S. pyogenes S. aureus Coag.neg.Staph C. neoformans Others non viable Total % Tables 3.2 and 3.3 show the distribution of the bacterial species isolated from CSF according to the age of the patient and the age-specific incidence per 100,000, respectively. Streptococcus agalactiae is still the predominant species isolated in neonates (younger than 1 month), and represented 69% of the isolates in this age group, whereas in the age group 1-11 months the predominant species were S. pneumoniae and N. meningitidis (together 79%). Since the introduction of the vaccine against H.influenzae type b, the number of cases of H.influenzae meningitis in the age group 0-4 year has strongly decreased (1992: 231; 2004: 17; 2005: 20; 2006: 13). Table 3.2 Isolates from CSF grouped according to patients' age, 2007 AGE AGE (YEARS) (MONTHS) SPECIES Total % N. meningitidis H. influenzae S. pneumoniae E. coli S. agalactiae L. monocytogenes S. pyogenes S. aureus Coag.neg.Staph C. neoformans Others non viable Total %

19 As anticipated from table 3.2, the incidence of bacterial meningitis was highest in the age group of 0-4 years (table 3.3), but this figure was lower than in the previous years (2004: 19.8; 2005:17.0; 2006: 14.27) Table 3.3 Age-specific incidence of bacterial meningitis per 100,000 inhabitants grouped according to species, 2007 AGE (YEARS) ISOLATE Total N. meningitidis H. influenzae S. pneumoniae E. coli S. agalactiae L. monocytogenes S. pyogenes S. aureus Coag.neg.Staph C. neoformans Others non viable Total Table 3.4 shows the frequency of the isolates per species from CSF according to gender of the patients. For most species the Male/Female ratio varied between 1.0 and 2.3. All S. pyogenes patients were female. Table 3.4 Isolates from CSF according to patients' gender, 2007 SPECIES M F M/F-ratio sex not known Total % N. meningitidis H. influenzae S. pneumoniae E. coli S. agalactiae L. monocytogenes S. pyogenes S. aureus Coag.neg.Staph C. neoformans Others non viable Total %

20 4 NEISSERIA MENINGITIDIS 4.1 General features In 2007, the Reference Laboratory received 171 Neisseria meningitidis isolates, of which 89 were isolated from CSF (or CSF and blood) (89 in 2006) and 82 from blood only (79 in 2006). This means that 48% of cases of meningococcal disease concerned patients with a positive blood culture only, either because no meningitis was present or because no CSF specimen was obtained. The distribution of isolates according to month of receipt shows that the highest number of isolates was received, as in most previous years, in the first quarter of the year (figure 4.1). Number of isolates 100 Isolates from blood only CSF or CSF and blood isolates Years Figure 4.1 Seasonal distribution of meningococcal disease,

21 4.2 Antibiotic susceptibility Ninety-eight percent of all isolates (167/171) were susceptible to penicillin (MIC 0.06 µg/ml; CSF isolates 97%, isolates from blood only 99%) (table 4.1). All isolates were susceptible to rifampicin. Table 4.1 Susceptibility* of N. meningitidis CSF and/or blood isolates to penicillin, 2007 Susceptibility to penicillin CSF Blood Total % MIC < MIC MIC > Total number of isolates % * MIC values in µg/ml 4.3 Serogroups Serogroup B accounted for 85% (2006: 92%) of all isolates and group C for about 5% (table 4.2). The remaining 10% of the isolates were of the rare serogroups X, Y, W135 or nongroupable. Remarkable is the increase of serogroup Y to 6% of all isolates. The serogroup distribution observed during the whole collection period (figure 4.2) shows that in 2007 the number of group B isolates (146 cases) was the lowest since The proportion of group C isolates was 24% in 1991, decreased to about 10% in 1994 and was since then increasing, with a sharp rise from 19% (105 cases) in 2000 to 40% (276 cases) in 2001 (figure 4.2). In June 2002, vaccination against serogroup C was included in the National Immunization Program. Since then, the number of serogroup C isolates received by the Reference Laboratory decreased rapidly to only a few isolates per year; in 2007 only 9 serogroup C isolates were received (figure 4.3). However, it should be noted, that also the number of serogroup B has decreased, albeit that the decrease started earlier and was less steep. 20

22 Number of isolates Serogroup A Serogroup B Serogroup C Years Figure 4.2. Distribution of meningococcal serogroups, Number of isolates Serogroup B Serogroup C Months Figure 4.3 Bimonthly distribution of meningococcal serogroups B and C,

23 4.4 Serogroup and age The age distribution of patients with meningitis and/or meningococcemia shows that 43% (74 of 171) of the patients was younger than 5 years (table 4.2, figure 4.4). Among patients from whom meningococci were isolated from blood only, 46% was younger than 5 years. Table 4.2 Serogroups of N. meningitidis (all isolates: from CSF and /or blood, absolute numbers) by patient age, 2007 AGE ( MONTHS) AGE (YEARS) SEROGROUP Total % B C X Y W Total % *n.g : non groupable Table 4.3 Serogroups of N. meningitidis (isolates from CSF, or CSF and blood; absolute numbers) by patient age, 2007 AGE ( MONTHS) AGE (YEARS) SEROGROUP Total % B C X Y W Total % Table 4.4 Age distribution of meningitis (incidence per 100,000 inhabitants) by different serogroups of N. meningitidis (isolates from CSF, or CSF and blood), 2007 AGE (YEARS) SEROGROUP Total B C X Y W

24 Total Table 4.5 Serogroups of N. meningitidis (isolates from blood only, absolute numbers) by patient age, 2007 AGE ( MONTHS) AGE (YEARS) SEROGROUP Total % B C X Y W Total % Table 4.6 Age distribution of meningococcemia (incidence per 100,000 inhabitants) by different serogroups of N. meningitidis (isolates from blood only), 2007 AGE (YEARS) SEROGROUP Total B C X Y W Total

25 4.5 Group B meningococci Figure 4.4 shows the age distribution of group B meningococcal disease. The age-specific incidences per 100,000 inhabitants in the age groups younger than 5 years and years were 7.2 and 1.6, respectively (2006: 7.0 and 2.0, respectively). The age-specific incidences in the age groups between 25 and 85 years were less than 0.6. Number of isolates N. meningitidis group B 80 N. meningitidis group B per 100,000 inhabitants 70 Number of isolates per 100,000 inhabitants Age group (years) 0 Figure 4.4 Age distribution of serogroup B meningococcal disease in

26 4.6 Distribution of PorA genosubtypes among serogroup B and C meningococci The monoclonal antibodies used for (sub)typing of meningococci are no longer available. Therefore, from January 1, 2005 on, typing of meningococcal isolates using monoclonal antibodies is not performed anymore by the Reference Laboratory. Instead, epitopes of PorA and FetA are determinded by sequencing of their DNA coding regions. The epitopes of PorA that react with the monoclonal antibodies of the subtyping scheme are encoded by the variable regions VR1 and VR2 of pora, encoding the outer membrane protein PorA. Since 2000 we routinely sequence the DNA regions which encode VR1 and VR2 of PorA of all meningococcal isolates. The DNA sequences are translated into putative amino acid sequences, which are then compared with the PorA epitopes present in the database available on the website: In 2007, 39 different VR1/VR2 combinations were encountered among serogroup B meningococci (2000: 73; 2001: 75; 2002: 71; 2003: 67; 2004: 60; 2005: 52; 2006: 50) The proportion of the dominant PorA genosubtype P1.7-2,4 decreased from 40% in 2000 to 27% in 2007 (figure 4.5; table 4.7). The nine serogroup C isolates had the VR1/VR2 combination 1.5,2 (4 times); 1.5-1,10-1; 1.5-1,10-8 (3 times) and 1.5-1,2-2, respectively. P1.7-2,4 27% P1.5-2,10 7% P1.7-2,13-2 7% P1.22,14 8% Other 39% P1.22,9 6% P1.19,15 3% P1.21,16 3% Figure 4.5 Distribution of group B meningococcal PorA types,

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28 Table 4.7 N. meningitidis serogroup B isolates according to PorA genosubtype, YEAR VR1,VR2 combination No. % No. % No. % No. % Vaccination types* 1.5-1, , , , , , , , , , , , , Non vaccination types 1.5, , , , , , , , , , Other Vaccination types Non vaccination types Total *based on a hexavalent PorA vaccine as proposed by P. van der Ley et al. Vaccine 1995;13:

29 4.7 Distribution of FetA genosubtypes among serogroup B and C meningococci In addition to sequencing of PorA epitopes, meningococcal isolates are also characterized by sequencing of an epitope of FetA. This outer membrane protein is involved in iron uptake by meningococci and is considered as a potential vaccine component. Therefore, the variability of this protein has been investigated intensively. The most variable part of the protein, called VR, has been used to establish a typing scheme. Analogous to PorA typing, the VR part of feta is sequenced and translated to a putative aminoacid sequence. So far, about 270 VR sequences comprising 6 classes, are identified, available at As an example of a type designation: F5-2, in which the first digit indicates the class and the second digit the variant of this class. In 2007, 22 different FetA variants were observed among serogroup B meningococci. The dominant type is F1-5, accounting for 40% of group B meningococci (figure 4.6; table 4.8). This FetA type is strongly linked with PorA VR1/VR2 P1.7-2,4 and P1.7-2,13-2 and the MLST clonal complex ST41/44. Of the 58 (68 in 2006) meningococci with the F1-5 FetA type, 34 (40 in 2006) were of P1.7-2,4 and 5 (3 in 2006) were of P1.7-2,13-2). The nine serogroup C meningococci had the FetA type F5-1 (1), F5-5 (4) and F3-6 (4). FetA type F1-5 40% FetA type F1-7 8% FetA type F3-3 8% FetA type F5-1 13% FetA type F5-5 10% Other 18% FetA type F5-12 3% Figure 4.6 Distribution of group B meningococcal FetA genosubtypes,

30 Table 4.8 N. meningitidis serogroup B isolates according to FetA genosubtype, FetA type YEAR No. % No. % No. % No. % F F F F F F F F F F F F Other Total

31 5 HAEMOPHILUS INFLUENZAE In total, 115 Haemophilus isolates were submitted to the Reference Laboratory. This number is comparable to that of the last years (table 2.3, figure 3.3). Nineteen strains were isolated from CSF (or CSF and blood), and 96 from blood only (2006: 25 and 95; 2005: 37 and 95; 2004: 32 and 93, respectively). Twenty-one (20.9%) of the isolates were H. influenzae type b (table 5.1). From 1999 to 2004, the number of H. influenzae type b isolates received by the Reference Laboratory increased (table 5.4). The higher number of H. influenzae type b isolates was mainly due to an increase of H. influenzae type b cases among elderly people. Since 2004 the number of H. influenzae type b isolates decreased with 50% to a total number of 24 in 2006 and Four cases of H. influenzae type b invasive disease were observed among children younger than 2 years of age (5 in 2006 and 15 cases in 2005) (figure 5.1). In total 87 non-typable H.influenzae were received; 9 isolated from CSF (or CSF and blood) and 78 isolated from blood only (table 5.1, 5.2 and 5.3). Except patients in age group 04 yr, non-typable strains were isolated more frequently than type b isolates (table 5.1). Table 5.1 Total number of H.influenzae isolates from CSF and/or blood, according to serotype and age, 2007 AGE (MONTH) AGE (YEAR) TOTAL TYPE Total % b e f n.t.* Total % * non-typable Table 5.2 H.influenzae isolates from CSF (or CSF and blood), according to serotype and age, 2007 AGE (MONTH) AGE (YEAR) TOTAL TYPE Total % b e f n.t.* Total % * non-typable 30

32 Table 5.3 H. influenzae isolates from blood only, according to serotype and age, 2007 AGE (MONTH) AGE (YEAR) TOTAL TYPE Total % b e f n.t.* Total % * non-typable Number of isolates Year Age group 0-1 (months) Figure 5.1 Age distribution of H. influenzae type b invasive disease in the first two years of life,

33 The proportion of non-typable isolates increased from 6% in 1992 to about 70% in the period (table 5.4). Table 5.4 H. influenzae isolates from CSF and/or blood received from 1992 to 2007 according to year and serotype SEROTYPE TOTAL CSF (or CSF YEAR a b d e f n.t.* Total % n.t.* and blood) * non-typable Blood only The absolute number of non-typable isolates from CSF remained stable during the period 1992 to 2006 as shown in table 5.5. In 2007 the number of non-typable isolates from CSF decreased to 9. The number of non-typable H. influenzae isolates from blood increased during during the period 1992 to 2007 from 15 to 78 in This may reflect an increase in the number of cases with invasive disease due to non-typable H.influenzae, but on the other hand may be due to an increased awareness of the importance of the results of serotyping. In addition, the sensitivity of blood cultures may be increased by the introduction of automated blood culture devices. The number of non-typable H.influenzae remained stable during the last four years. 32

34 Table 5.5 Non-typable H. influenzae isolates from CSF and/or blood received from 1992 to 2007 YEAR non typable CSF or CSF and blood Blood alone The proportion of ß-lactamase producing invasive H. influenzae isolates (CSF and/or blood) is decreasing since % of isolates Years Figure 5.2 Percentage -lactamase producing H. influenzae,

35 6 STREPTOCOCCUS PNEUMONIAE 6.1 General features The Reference Laboratory received 1005 S. pneumoniae isolates. Of these, 243 were isolated from CSF or from CSF and blood (table 2.3; figure 6.1). The incidence of pneumococcal meningitis slightly rose since 1990 from 1.0 to 1.6 in 2004; subsequently it slightly decreased to 1.3 in In 2007 the incidence was 1.5. A steep increase in the number of pneumococcal blood isolates had occurred between 1994 (312 isolates) and 2003 (1471 isolates). This increase can be explained by the increasing use of automated blood culture devices by the contributing laboratories and by a real increase in the number of cases of pneumococcal bacteremia due to pneumonia among patients of the increasing cohort of the elderly (figure 6.1) and a more complete submission of isolates by the laboratories. The number of isolates from blood sent to the Reference Laboratory decreased from 1471 in 2003 to 762 in This was due to a change in policy: from 2003 onwards, we asked only nine selected laboratories, evenly distributed over the country, to send pneumococcal blood isolates. Thus, the numbers of S. pneumoniae from blood only are incomplete. This policy has been changed to monitor the effect of the introduction of the 7-valent conjugate pneumococcal polysaccharide vaccine by June 1 st, From 2006 onwards, all laboratories are requested to send all invasive pneumococcal isolates from patients in the age group 0-4 year, while from patients older than 4 year only isolates from CSF are requested. Again, from nine selected laboratories we ask all invasive pneumococcal isolates from all patients. Number of isolates Per 100,000 inhabitants S.pneumoniae from CSF or CSF and blood S.pneumoniae from blood only S.pneumoniae from blood only from 9 selected labs S.pneumoniae from CSF or CSF and blood from 9 selected labs S.pneumoniae from CSF or CSF and blood, per 100,000 inhabitants Years 0 Figure 6.1 Distribution of S. pneumoniae isolates,

36 6.2 Antibiotic susceptibility Among 243 isolates from CSF (or CSF and blood) and 762 isolates from the blood only, 1 (0.4%) and 12 (1.6%), respectively, were not susceptible to penicillin (MIC > 0.06 mg/l, table 6.1). Only 4 (0.5%) from blood only strains were resistant to penicillin (MIC > 1.0 mg/l). Table 6.1 Susceptibility* of S. pneumoniae isolates to penicillin, 2007 penicillin Isolate MIC < MIC 1.0 MIC >1.0 Total % CSF or CSF and blood Blood only Total number of isolates % * MIC values in µg/ml Figure 6.2 shows the distribution of S. pneumoniae isolates according to the patients age. The incidence of pneumococcal meningitis is highest among children in their first year of life. A second peak of high incidence was observed in the age group year (Table 6.5). Number of isolates S.pneumoniae isolated from CSF or CSF and blood S.pneumoniae isolated from blood only S.pneumoniae isolated from CSF or CSF and blood, per 100,000 inhabitants Per 100,000 inhabitants Age group (years) Figure 6.2 Distribution of S. pneumoniae isolates received in 2007 according to age 35

37 6.3 Distribution according to serotype The serotype distribution of CSF (or CSF and blood) isolates is shown in table 6.2. Table 6.2 S. pneumoniae serotype of isolates from CSF, 2007 TYPE Number TYPE No. 1 of isolates B C C 17 4 C 5 19 F C 11 6 A 7 19 A 9 6 B C F F A 1 9 N 4 23 F C 22 9 V C A 3 10 A 8 23 B 2 11 A 4 24 F 3 12 F C A 1 33 F 5 15 B C 1 35 F 2 16 F 2 35 B 1 17 F 1 C Total 243 type represented in the 23-valent pneumococcal vaccine (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, 33F) type represented in the 7-valent conjugated vaccine 36

38 Table 6.3 S. pneumoniae isolates from CSF and/or blood, by serotype and age of patients, 2007 AGE ( MONTHS) AGE (YEARS) TYPE Total % Others Total % The relationship between age and major types of all isolates is shown in table 6.3. For isolates from CSF (or CSF and blood) only, the distribution of serotypes by age of the patient is presented in table 6.4, while the incidence of S. pneumoniae meningitis per serotype per 100,000 inhabitants is shown in table 6.5. The distribution of serotypes by age of the patient for pneumococcal isolates from blood only is shown in tables 6.6. As aforementioned, incidences of S. pneumoniae from blood only are incomplete. Table 6.7 show the distribution of CSF isolates according to serotype over the last ten years. Table 6.4 S. pneumoniae isolates from CSF (or CSF and blood), by serotype and age of patients, 2007 AGE ( MONTHS) AGE (YEARS) TYPE Total % others Total %

39 Table 6.5 Age-specific incidence of pneumococcal meningitis (isolates from CSF or CSF and blood) per 100,000 inhabitants according to type, 2007 AGE (YEAR) TYPE Total Others Total Table 6.6 S. pneumoniae isolates from blood only, by serotype and age of patients, 2007 AGE ( MONTHS) AGE (YEARS) TYPE Total % others Total %

40 Table 6.7 Distribution of pneumococcal CSF isolates according to serotype, TYPE A B F C A N V A F A F A A B C F F F A B C F A F A F A B F A F F A F B Rough (n.t.) Total Year Bold: type represented in the 23-valent pneumococcal vaccine (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, 33F) ItalicUnderlined: type represented in the 7-valent conjugated vaccine. 39

41 7 ESCHERICHIA COLI The Reference Laboratory received 27 Escherichia coli strains, 15 isolated from CSF (or CSF and blood) and 12 from blood only (table 7.1, 7.2 and 7.3). The number of E. coli isolates from CSF was two times higher than that in the last year. (2006: 6; 2005: 11; 2004: 8; 2003: 15; 2002: 17). Only 47% of the cases of E. coli meningitis occurred in the first month of life, while 75% of the patients with E. coli sepsis were younger than one month. Interestingly, the types O4, O6, O11, O16, O44, O73, O99 and O-non-typable are prevalent among non-k1 isolates, while the types O2, O7, O12, O18, O21 and O134 are more often found among K1 isolates. O16 and O-non-typable were found in both K groups. Table 7.1 Serotypes of E. coli isolates from CSF and/or blood, by age of patients, 2007 AGE (MONTH) AGE (YEAR) TOTAL TYPE Total % Non K K Total % Table 7.2 Serotypes of E. coli isolates from CSF (or CSF and blood), by age of patients, 2007 AGE (MONTH) AGE (YEAR) TOTAL TYPE Total % Non K K Total % Table 7.3 Serotypes of E. coli isolates from blood only by age of patients, 2007 AGE (MONTH) AGE (YEAR) TOTAL TYPE Total % Non K K Total %

42 41

43 8 STREPTOCOCCUS AGALACTIAE (group B) In 2007 the number of Streptococcus agalactiae isolates received by the Reference Laboratory was almost 20% higher than last years (2007: 58; 2006: 47; 2005: 46; 2004: 50). Twenty-six S.agalactiae isolates were from CSF (or CSF and blood) and 32 from blood only (table 8.1, 8.2 and 8.3). About 86% of the cases occurred in the first month of life. Serotype III was the most prevalent (table 8.1). Table 8.1 Serotypes of S. agalactiae isolates from CSF and/or blood, by age of patients, 2007 AGE (MONTH) AGE (YEAR) TOTAL TYPE Total % Ia Ib II III V VI Total % Table 8.2 Serotypes of S. agalactiae isolates from CSF (or CSF and blood), by age of patients, 2007 AGE (MONTH) AGE (YEAR) TOTAL TYPE Total % Ia Ib II III V VI Total %