Scholarly article on topic 'Genome-based study of a spatio-temporal cluster of invasive meningococcal disease due to Neisseria meningitidis serogroup C, clonal complex 11'

Genome-based study of a spatio-temporal cluster of invasive meningococcal disease due to Neisseria meningitidis serogroup C, clonal complex 11 Academic research paper on "Agriculture, forestry, and fisheries"

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Abstract of research paper on Agriculture, forestry, and fisheries, author of scientific article — P. Stefanelli, C. Fazio, A. Neri, A. Ciammaruconi, E. Balocchini, et al.

Summary Objectives To describe a spatio-temporal cluster of invasive meningococcal disease (IMD) due to serogroup C meningococci, occurred in a restricted area of Tuscany between January and October 2015, and the results of whole genome sequencing (WGS). Methods Surveillance activities and public health measures were implemented in the Region. Bacterial isolates from IMD cases were characterized by the National Reference Laboratory of the Istituto Superiore di Sanità (ISS), and WGS was performed on available strains. The kSNP software was used to identify core genome SNPs. Results Overall, 28 IMD cases due to meningococcus C were identified up to 31st October, 2015. Of them, 26 were due to meningococcus C:P1.5-1,10-8: F3-6:ST-11 (cc11) and 2 to C:P1.5-1,10-8: F3-6:ST-2780 (cc11). WGS of 13 meningococci isolated during the outbreak occurred in Tuscany in 2015 showed higher similarity when compared with those of 47 C: P1.5-1,10-8: F3-6:ST-11 (cc11) invasive strains from sporadic cases previously detected in Italy. Conclusions A highly aggressive meningococcal C strain was involved in the cluster of severe IMD occurred in Tuscany, a Region with high vaccine coverage among children. Whether this was due to low herd immunity related to the short duration of vaccine protection needs further investigation.

Academic research paper on topic "Genome-based study of a spatio-temporal cluster of invasive meningococcal disease due to Neisseria meningitidis serogroup C, clonal complex 11"

Journal of Infection (2016) xx, 1-9

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British Infection Association

www.elsevierhealth.com/journals/jinf

Genome-based study of a spatio-temporal cluster of invasive meningococcal disease due to Neisseria meningitidis serogroup C, clonal complex 11

P. Stefanellia *, C. Fazio a h, A. Neria h, A. Ciammaruconib,

E. Balocchinic, A. Anselmo b, C. Azzarid, G.M. Rossolinie,f g, P. Vacca a, A. Fortunato b, A. Palozzib, S. Fillo b, F. Lista b, M. Moriondo d, F. Nieddu d, G. Rezzaa

Q2 a Department of Infectious, Parasitic & Immuno-mediated Diseases, Istituto Superiore di Sanita, Rome, Italy

bMolecular Biology Section, Army Medical and Veterinary Research Center, Rome, Italy c Living Environment, Food and Veterinary Prevention and Safety Office, Tuscany Region, Florence, Italy

d Laboratory of Immunology and Infectious Diseases, Anna Meyer Children's University Hospital, University of Florence, Florence, Italy

e Department of Medical Biotechnologies, University of Siena, Italy f Department of Experimental and Clinical Medicine, University of Florence, Italy gMicrobiology and Virology Unit, Florence Careggi University Hospital, Italy

Accepted 16 May 2016 Available online ■ ■ ■

KEYWORDS

Meningococci; Neisseria meningitidis; Surveillance; Whole genome sequencing

Summary Objectives: To describe a spatio-temporal cluster of invasive meningococcal disease (IMD) due to serogroup C meningococci, occurred in a restricted area of Tuscany between January and October 2015, and the results of whole genome sequencing (WGS). Methods: Surveillance activities and public health measures were implemented in the Region. Bacterial isolates from IMD cases were characterized by the National Reference Laboratory of the Istituto Superiore di Sanita (ISS), and WGS was performed on available strains. The kSNP software was used to identify core genome SNPs.

* Corresponding author. Department of Infectious, Parasitic & Immune-mediated Diseases, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy. Tel.: +39 06 49902126; fax: +39 06 49387112. E-mail address: paola.stefanelli@iss.it (P. Stefanelli). h These authors contributed equally to this article.

http://dx.doi.org/10.1016Zj.jinf.2016.05.003

0163-4453/© 2016 The Authors. Published by Elsevier Ltd on behalf of The British Infection Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Results: Overall, 28 IMD cases due to meningococcus C were identified up to 31st October, 2015. Of them, 26 were due to meningococcus C:P1.5-1,10-8: F3-6:ST-11 (cc11) and 2 to C:P1.5-1,10-8: F3-6:ST-2780 (cc11). WGS of 13 meningococci isolated during the outbreak occurred in Tuscany in 2015 showed higher similarity when compared with those of 47 C: P1.5-1,10-8: F3-6:ST-11 (cc11) invasive strains from sporadic cases previously detected in Italy. Conclusions: A highly aggressive meningococcal C strain was involved in the cluster of severe IMD occurred in Tuscany, a Region with high vaccine coverage among children. Whether this was due to low herd immunity related to the short duration of vaccine protection needs further investigation.

© 2016 The Authors. Published by Elsevier Ltd on behalf of The British Infection Association. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Italy is considered a country at low incidence of invasive meningococcal disease (IMD), with predominance of the meningococcal B and C capsular serogroups.1 However, IMD cases due to serogroup C (MenC) declined after vaccine introduction2 consistently with the trends observed in other EU countries.3-7 In Italy, the use of the meningococcal C conjugate (MCC) vaccine for children 13-to-15 month olds and for adolescents 11-to-18 year-olds was recommended by the Italian National Immunization Plan for the years 2012/2014.8 However, few Regions, like Tuscany, introduced the MCC vaccine in the Regional immunization schedule since 2005. Initially, the regional plan provided three doses at 3, 5, and 13 months of age, switching to a single dose at 13 months, with a catch-up immunization until 6 years of age, in 2008.

In some Regions, a booster dose with the conjugate vaccine against meningococcal A, C, W and Y was provided to adolescents, people at risk, and travelers to endemic areas.

A wide variation in vaccination coverage between Italian Regions was observed, with values ranging between 42.72% and 88.28%; in Tuscany the coverage was around 87.2% in the birth-cohort of the year 2012 (http://www.salute.gov.it/ imgs/C_17_tavole_20_allegati_iitemAllegati_2_fileAllegati_ itemFile_0_file.pdf). Unfortunately, data on immunization coverage for teens and at-risk groups were not available.

Despite the extended use of the vaccine, outbreaks and smaller clusters of MenC belonging to the ST-11 clonal complex (cc11) (MenC:cc11) have continued to occur in Europe; in particular, the finetype C:P1.5-1,10-8:F3-6:ST-11 (cc11) was responsible of outbreaks in France and in Germany.9'10

Since meningococci belonging to cc11 are considered highly virulent and able to cause outbreaks, it is important to identify and characterize this aggressive, vaccine preventable strain. Hereby, we describe a cluster of cases of IMD due to MenC:cc11 occurred between January and October 2015 in Tuscany. In particular, we describe the epidemic dynamics and the characteristics of the strain involved in the outbreak up to that date. To this end, the results of the whole genome sequencing (WGS), and the geo/temporal distribution and characterization of the isolates compared with other strains of the same

finetype identified in different geographical areas are presented.

Materials and methods IMD surveillance and control

In Italy, IMD cases are mandatorily reported, through the Regions, to the Ministry of Health and to the Italian Institute of Public Health (Istituto Superiore di Sanita, ISS). The EU case-definition for lab-confirmed cases is utilized.11 When a case of IMD is identified, contact tracing and chemoprophylaxis are provided by the local health authorities. In Tuscany, after the detection of an increase in the number of IMD cases, a number of actions were undertaken. Public health measures consisting in early detection (i.e. 24 h rapid diagnosis using molecular methods) and treatment of cases, antibiotic prophylaxis of close contacts, active offer of the tetravalent vaccine to all teenagers (up to 20 years of age), and free-of-charge vaccination of the adults (up to 45 years) living in the areas at greatest risk. We defined an "high risk area" a province with an observed number of cases in 2015 (up to October 31st) at least two-fold higher than expected (the number of cases reported in the same province in the last 5 years).

The microbiological characterization of invasive menin-gococcal strains was performed by the National Reference laboratory (NRL) of the ISS. Epidemiological, clinical, and microbiological data for each IMD case were managed using a dedicated database.

Microbiological analyses

Isolates were cultured following standard procedures. The serogroup was confirmed by slide agglutination with commercial antisera (Remel Europe, Ltd, UK) or by multiplex PCR.12 Susceptibility to cefotaxime, ceftriaxone, ciprofloxa-cin, penicillin G, and rifampicin was determined by the MIC Test Strip Method (Liofilchem, Italy) on Mueller-Hinton agar (Oxoid), supplemented with 5% of sheep blood. The breakpoints are those recommended by the European Committee on Antimicrobial Susceptibility Testing — EUCAST version 5.0, January 1, 2015 (http://www.eucast.org/).

Genomic DNA was extracted using the QiAmp mini kit (Qiagen, Hilden, Germany), according to the

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manufacturer's instructions, from an overnight culture grown on Thayer Martin agar plate or directly from the clinical sample, blood, or CSF (cerebrospinal fluid).

Multilocus sequence typing (MLST), PorA and FetA typing, Bexsero antigen genes and antibiotic resistance genes were defined as described in http://neisseria.org/. The finetype is identified as follows: capsular group: porA (P1). VR1,VR2: fetA VR: ST (cc).

Whole genome sequencing

The whole genome sequencing was performed on 60 Neisseria meningitidis isolates at the Army Medical and Veterinary Research Center in Rome.

For each isolate, 1 ng of DNA was used to prepare the sequencing libraries, following the Nextera XT DNA protocol, according to the manufacturer's instructions. WGS of the isolates was performed by using the Illumina MiSeq platform (kit v3, 600 cycles). On average, 1.9 million paired-end reads were obtained for each sample. A first quality check of the raw sequence data was performed using FastQC.13 Reads were trimmed to keep high quality bases (Q score >25) using the software Sickle,14 and de novo assembly was carried out with the ABySS software version 1.5.2 (k parameter = 63).15 Contigs longer than 500 bp were selected using an ad hoc script and kept for further analysis. The final assembly ranged from 201 to 506 (median = 247) contigs/sample covering the ~2.2 Mb of the N. meningitidis genome.

Genome comparison

Draft genomes were uploaded to the PubMLST.org database (http://pubmlst.org/neisseria/), which runs on the BIGSdb platform.

Genomes were analyzed and compared using the BIGSdb Genome Comparator tool implemented within the PubMLST website (http://pubmlst.org/neisseria/), through the gene-by-gene analysis approach. Phylogenetic analysis of the 60 isolates was performed firstly exploring the 53 ribo-somal protein (rMLST) genes. Then, the genomes were investigated at higher resolution, analyzing the 1605 loci, defined as the core genome in the PubMLST Neisseria database (http://pubmlst.org/neisseria/), by the core genome MLST (cgMLST) approach.

Incomplete loci were automatically removed from the distance matrix calculation for the Neighbor-Net graphs. The resulting distance matrices were visualized as Neighbour-net networks, generated by SplitsTree4 (version 4.13.1).16

SNPs analysis

The program kSNP17 was used to identify core genome SNPs on WGS data for each isolate. kSNP is based on k-mer analysis, therefore no multiple sequence alignment is required. Before running kSNP, the program Kchooser was used to estimate the optimum value of k-mer that for N. meningitidis data set was 31. After the kSNP analysis, the core_SNP_ma-trix output file, containing only SNP loci common to all isolates, was used for further analysis. Maximum Likelihood

analysis18 on the core SNP matrix output of kSNP was performed in MEGA 6.06,19 using the Bootstrap Method with 100 Bootstrap replications.

Results

Epidemiological findings

From January 1st to October 31st 2015, 34 IMD cases were detected in Tuscany; of them, 28 were due to MenC. IMD cases were reported almost every week (Fig. 1) from towns and villages between Florence and Pisa. No secondary cases were identified. As already mentioned, traditional measures, including chemoprophylaxis of close contacts and vaccination of adolescents and adults, were promoted by Local Health Authorities to contain the spread of the disease. In particular, a catch-up strategy was used for unvaccinated adolescents, whereas booster doses were administered to those who had been previously vaccinated.

In the first 10 months of 2015, the incidence of IMD due to MenC in Tuscany was around 0.7 per 100,000 inhabitants, which is almost 10-fold higher than expected for the entire year in Tuscany since the introduction of MCC vaccination in 2005 (data not shown).

The median age of the patients was 25.5 years (average: 31 years), ranging from 9 to 82 years; 15 patients were females and 13 males. Meningitis and septicaemia represented the main clinical picture (14 cases), followed by septicaemia alone (13 cases), whereas only one patient presented meningitis without sepsis. The outcome was fatal for 6 patients who developed septicaemia or meningitis/ septicaemia.

Two patients had received the MCC vaccine 8 years before the onset of the disease, and one patient 2 years before; all the other patients were unvaccinated. The NRL received samples from 26 of 28 IMD cases: 12 strains, 2 bacterial suspensions, and 12 clinical samples (CSF or blood). All the 12 strains were susceptible to cefotaxime, ceftriaxone, ciprofloxacin, and rifampicin, but showed a decreased susceptibility to penicillin G (range 0.094-0.25 mg/L).

Molecular analyses

Molecular analysis was performed on all 26 available samples. MLST identified the ST-11 complex (cc11) as the unique clonal complex. However, among cc11 isolates, two different sequence types (STs) were found: ST-11 (24 samples) and ST-2780 (2 samples, ID 2670 and 2691). The ST-2780 was identified in 2 strains isolated in April and May, and differed from the ST-11 for 15 nucleotide in the fumC gene.

Two different finetypes were identified: C: P1.5-1,10-8: F3-6: ST-11 (cc11) in 24 samples (92.3%), and C: P1.5-1,10-8: F3-6: ST-2780 (cc11) in 2 samples (7.7%).

The fHbp-1.13 variant was found in all but one analyzed strains (ID 2639) that presented a fHbp allele 669, encoding a truncated fHbp protein due to a frameshift mutation.

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months and days

Figure 1 Timeline of serogroup C IMD cases occurred in Tuscany from January to October 31st 2015.

All meningococci showed the NHBA-20 variant and NadA with the Insertion Sequence (IS) disrupting the gene, as already described for the ET-15 meningococci.20

As already described,21 a premature stop codon in the aniA gene was also identified in all the samples.

Genome comparison

Whole genome analysis

Whole genome analysis was performed on 58 C: P1.5-1,10-8: F3-6: ST-11 (cc11) strains and two C: P1.5-1,10-8: F3-6: ST-2780 (cc11) collected in Italy from 2007 to 2015; out of these strains, 6 were from an outbreak occurred in Veneto in 2007, 4 from an outbreak occurred on a cruise ship in Tuscany in 2012, and 13 from the current outbreak (Tuscany, 2015); the remaining 37 were from sporadic IMD cases.

The analysis of 60 N. meningitidis isolates identified 9 ribo-somal sequence type (rST). All isolates belonging to the outbreak occurred in Tuscany in 2015 and 14 isolates collected from sporadic cases in Italy between 2013 and 2015 grouped together in the same rST (data not shown).

cgMLST

Overall, 1349 of the 1605 core genome loci were included in the cgMLST analysis, while the remaining 256 loci were incompletely assembled. A total of 653 genes resulted identical in all the 60 isolates.

The 60 meningococci were resolved by cgMLST analysis (Fig. 2, panel a), revealing four groups: i) group I, comprising 12 out of 13 meningococci identified in Tuscany in 2015 and 14 meningococci isolated from sporadic cases in Italy in 2013, 2014, and 2015, respectively (Fig. 2, panel b); ii) group II, including 4 strains responsible of an outbreak occurred on a cruise ship in 2012 in Tuscany22; iii) group III, comprising the outbreak occurred in Veneto in 200723; iv) group IV, including 1 strain isolated in Tuscany in 2015 (ID 2639) and 20 isolated from sporadic cases occurred in Italy from 2012 to 2015. Three isolates from sporadic IMD cases resulted distinct from the four groups.

Group I strains shared 1247 genes; in particular, those strains isolated in Tuscany in 2015 showed 1287 identical genes among them. The groups I and II, closer to each

other, shared 1157 exclusive identical genes. Groups III and IV were in distal regions and shared respectively 1044 and 765 genes with group I.

cgMLST analysis pointed out 11 genes specific for each group. For 5 of 11 genes, the function is known: NEIS0430, hypothetically coding for a cytoplasmic axial filament protein (cafA); the ctrE gene, involved in the capsule translocation, critical for transport of the mature lipidated polymers to the meningococcal cell surface24; the rpsD gene, coding for the 30S ribosomial protein S4; the tpsB gene, a hemolysin activator; the penA, coding for the penicillin-binding protein 2 (PBP2). The tpsB allele "new#1" harbors a deletion of 1 adenine in the polyA stretch, causing a premature stop codon 150 bp downstream.

Table 1 listed the allelic profiles of the 11 genes for the groups I and II, together with 3 representative genomes available in the website (www.neisseria.org, last access 22nd September 2015) and identified in UK. The genomes indicated in the Table 1 include those of meningococci Q1 belonging to C:P1.5-1,10-8:F3-6:ST-11 (cc11) with the penA 248 allele.

Concerning the 11 genes profile specific for each groups, the main findings were:

i) 10 out of the 11 genes were identical in the group I; NEIS0430 distinguished one subgroup within group I, including all the genomes obtained from strains isolated in Tuscany in 2015, and 4 from sporadic cases occurred in Italy; ii) genomes of the group II, obtained from the outbreak strains occurred in Tuscany in 2012, were identical to each other; in particular, the genomes obtained from these strains showed 3 genes (NEIS0430, ctrE, and rpsD) identical to the genome identified in UK ID21253; moreover, they showed also the NEIS1858 gene identical to the UK genome ID 30209; iv) the genomes obtained from strains isolated in Tuscany in 2015 were identical to the UK ID 21253 genome.

SNPs analysis

A Neighbor Joining analysis was performed on 60 N. menin-gitidis strains using 2885 core SNPs obtained by the kSNP analysis of WGS data (Fig. 3). All the strains involved in the cluster occurred in Tuscany in 2015, with the exception of the strain 2639, grouped together. The strain 2639 showed a unique SNP profile, similar to those of strains

Figure 2 Panel (a), Neighbour-net phylogenetic network based on a comparison of 1349 core genome loci (cgMLST) among 58 N. meningitidis C: P1.5-1,10-8: F3-6: ST-11 (cc11)and 2C: P1.5-1,10-8: F3-6: ST-2780 (cc11). Panel (b) Neighbour-net phylogenetic network based on a comparison of 1360 core genome loci (cgMLST) among 26 isolates of the group I; ID of isolates responsible for serogroup C IMD cases in Tuscany in 2015 are marked. The scale bars indicate the number of differences among the compared loci.

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Table 1 Allelic profiles of 11 genes characterizing groups I and II as defined by cgMLST analysis of meningococci isolated in Italy and of 3 representative meningococcal genomes available at http://pubmlst.org/Neisseria (last access 22nd of September 2015), selected by finetype C:P1.5-1,10-8:F3-6: ST-11 (cc11) and penA 248 allele.

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ID (ID http://pub3lst.org/Neisseria) Country Year Group in the cgMLST tree NEIS0430 ctrE rpsD tpsB NEIS0263 NEIS0264 NEIS0265 NEIS0745 NEIS1594 NEIS1858 penA

2539 (37600) Italy 2013 I 6 355 29 new#1 144 10 28 176 271 157 248

2493 (37594) Italy 2013 I 6 355 29 new#1 144 10 28 176 271 157 248

2541 (38867) Italy 2013 I 6 355 29 new#1 144 10 28 176 271 157 248

2460 (38847) Italy 2013 I 46 355 29 new#1 144 10 28 176 271 157 248

2560 (36461) Italy 2014 I 46 355 29 new#1 144 10 28 176 271 157 248

2530 (37597) Italy 2014 I 6 355 29 new#1 144 10 28 176 271 157 248

2531 (37598) Italy 2014 I 6 355 29 new#1 144 10 28 176 271 157 248

2555 (37601) Italy 2014 I 6 355 29 new#1 144 10 28 176 271 157 248

2662 (36766) Italy 2014 I 6 355 29 new#1 144 10 28 176 271 157 248

2569 (36765) Italy 2014 I 398 355 29 new#1 144 10 28 176 271 157 248

2632 (36771) Italy 2015 I 6 355 29 new#1 144 10 28 176 271 157 248

2636* (36448) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2644* (36450) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2652* (36451) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2654* (36386) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2656* (36769) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2664* (36452) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2665* (36453) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2669* (36402) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2670* (36844) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2691* (36767) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2701* (36784) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2718* (38839) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2646 (36770) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2690 (36768) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2717 (38838) Italy 2015 I 398 355 29 new#1 144 10 28 176 271 157 248

2417** (36444) Italy 2012 II 6 209 1 1 1 1 1 1 1 3 248

2418** (36445) Italy 2012 II 6 209 1 1 1 1 1 1 1 3 248

2419** (36446) Italy 2012 II 6 209 1 1 1 1 1 1 1 3 248

2420** (36447) Italy 2012 II 6 209 1 1 1 1 1 1 1 3 248

M11 241039 (21253) UK 2011 na 6 209 1 new#1 144 10 28 176 271 157 248

M13 240628 (30209) UK 2013 na 6 209 1 new#1 144 10 28 176 271 3 248

M13240155 (28093) UK 2013 na 6 355 29 new#1 144 10 28 176 271 157 248

'Tuscany 2015; ** Tuscany 2012; na: not applicable.

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1000000000099999999990000000000000000000077777777776666666 0900765432i0900765432i0900765432i0900765432i090076543

Figure 3 Maximum Likelihood-based tree of 58 N. meningitidis C: P1.5-1,10-8: F3-6: ST-11 (cc11) and 2 C: P1.5-1,10-8: F3-6: ST-2780 (cc11) constructed on 2885 core SNPs. ID of isolates responsible for serogroup C IMD cases in Tuscany in 2015 are marked with an asterisk.

from sporadic cases from other Italian Regions. The Between Groups Mean Distance was 0.038 between the Tuscany outbreak strains (Number core SNPs: 177) and the isolates 2691 and 2670, which showed a different Sequence Type (ST 2780).

The relatedness between the MenC strains isolated in Tuscany in 2015 and in the 2012 outbreak was supported by the number of base substitutions per site (Between Groups Mean Distance in MEGA6) of only 0.071.

Discussion

In a 10 months period, 28 IMD cases due to serogroup C within the cc11/ET-15 clonal complex were reported in a geographically restricted area of Tuscany. All but two isolates belonged to the finetype C:P1.5-1,10-8:F3-6:ST-11 (cc11). This strain had been already identified and characterized in several European countries,9,10 including two smaller outbreaks occurred in Italy.22,23

The number of IMD cases due to MenC reported in the first 10 months of 2015 in Tuscany was higher than expected. For comparison, in 2014, a total of 156 lab-confirmed IMD cases was reported in Italy, and only two were due to MenC; the majority of cases was due to

meningococcus B, as a consequence of the decline of serogroup C cases among children 0—5 years old, following the introduction of childhood vaccination. In Tuscany, the incidence of IMD cases due to MenC was 0.2 cases/100.000 inhabitants before the introduction of MCC (2000—2005), declining to 0.07 after the introduction of vaccination (2006—2014), then showing this unexpected peak of 0.7 in 2015. This was to some extent surprising, since the coverage of MCC vaccination in the Region was high.

Traditional measures, including chemoprophylaxis of close contacts and vaccination of adolescents and young adults, were implemented to contain the spread of IMD cases. In particular, a catch-up strategy, boosting adolescents who had been vaccinated and immunizing those who had not been vaccinated before, was first implemented. In fact, the lack of a catch-up program targeting adolescents and young adults, who are considered the principal responsible for carriage and transmission, is likely to explain at least in part the excess of IMD cases observed in Tuscany. Since several cases were reported among young adults, it was then decided to offer the vaccine to all unvaccinated individuals, up to 45 years old, living in the affected area. Up to October 31st 2015, about 180,000 individuals have been vaccinated (data not shown).

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8 P. Stefanelli et al.

Unfortunately, data on the prevalence of MenC carriage MenC carriage during outbreaks of MenC disease, a study 63

by age category during the outbreak were not available. from Brazil found relatively high rates.33 The same study 64

Thus, the vaccination strategy was based on the assumption suggested that the polysaccharide vaccine had no effect 65

of a rather short duration of protection among already on carriage, whereas the MCC was able to reduce it. Sec- 66

vaccinated infants and young children, and a rapid circu- ondly, information on risk factors was not routinely and sys- 67

lation of MenC strains among unvaccinated adolescents and tematically collected. Thus, circulation dynamics and 68

young adults.25,26 To this regard, the MCC vaccine is likely infection modalities in the affected population remain 69

to provide high levels of direct protection in the short largely undefined. However, the characteristics of the cases 70

term26,27 and to reduce the prevalence of serogroup car- suggest a widespread circulation of MenC:cc11 among un- 71

riage, improving the herd immunity in the overall popula- vaccinated age cohorts. For this reason, it was decided to 72

tion.28,29 However, in areas where children are the main vaccinate adolescents and young adults, who are likely to 73

vaccination target, a relatively short duration of protec- sustain MenC circulation and represent the population 74

tion, combined with low vaccine coverage in the adoles- where IMD cases were mostly concentrated. 75

cence, may explain the occurrence of IMD cases among In conclusion, our findings show that increased incidence 76

unvaccinated individuals in older age-groups. In this rates of IMD due to hypervirulent meningococcal strains 77

context, adolescents and young adults are known to have may occur, unpredictably, also in areas with high vaccina- 78

the highest N. meningitidis carriage rates.30 tion coverage among infants and young children. WGS 79

As mentioned above, the outbreak was caused by the supports the hypothesis that C:P1.5-1,10-8:F3-6:ST-11 80

hypervirulent clone C:P1.5-1,10-8:F3-6:ST-11 (cc11). To (cc11) is an hypervirulent, outbreak-associated, and 81

support the analysis of the epidemic dynamics, whole evolving strain, which is now spreading globally. Detailed 82

genome sequencing was performed to characterize the molecular information is key to better understand epidemic 83

Neisseria pathogenome, and to compare the genomic char- and hyper-endemic dynamics, and to re-modulate vaccina- 84

acteristics of the 2015 Tuscany outbreak strains with those tion strategies. 85

of meningococci of the same finetype isolated in Italy over 86

the last years. Our findings indicate that all the C:P1.5- 87

I,10-8:F3-6:ST-11 (cc11) strains with penA248 isolated in Funding 88 Italy show high similarity. This strain was identified for 89 the first time during the outbreak occurred on a cruise This work was partly funded by the Italian Ministry of 90 ship docking in the port of Livorno, Tuscany, in 201 2.22 Health-CCM Project "Sorveglianza delle malattie invasive 91 Whether this event led to the introduction and spread of da N. meningitidis, Streptococcus pneumoniae ed Haemo- 92 this strain is matter of debate. philus influenzae" 2014. 93

WGS may greatly contribute to outbreak investigation, 94

allowing the identification of specific lineages causing 95

disease in specific age-groups.31 Using conventional Conflict °f interest 96

methods able to characterize the finetype of each isolate, 97

strains collected from IMD cases occurred in Tuscany in The authors declare no conflict of interest. 98

2015 appear similar to the other strains with the same fine- 99

type, which were responsible of sporadic cases occurring 100

throughout the country over the years. WGS and, in partic- Transparency declaration 101

ular, core genome sequencing demonstrated that meningo- 102

cocci isolated in 2015 in Tuscany were closely related to PS reports research grant from Novartis Vaccines & Diag- 103

each other, whereas they differed from the strains sharing nostic (GSK) for unrelated project. PS and GR participated 104

the same finetype which had been isolated in other Italian in a scientific meeting sponsored by Novartis Vaccines & 105

Regions. The SNP analysis confirmed the findings of cgMLST, Diagnostic (GSK). Q3 106

identifying the two strains with the same finetype but a 107

different ST (ST-2780). 108

As already mentioned by Lucidarme et al.,32 there are Acknowledgments 109

several globally spread cc11 strains belonging to different lin- 110

eages. Our analysis suggests the relationship between the These data were made available via the Neisseria Multi 111

2015Tuscany outbreak strains and the cc11/ET15 sublineage Locus Sequence Typing website (http://pubmlst.org/ 112

II.2 described by Lucidarme et al.32 Interestingly, several neisseria) developed by Keith Jolley and sited at the Uni- 113 C:P1.5-1,10-8:F3-6:ST-11 (cc11) isolated in the UK from versity of Oxford (Jolley KA, Maiden M. BIGSdb: scalable 114 2011 to 201532 showed allelic profiles similar to those identi- analysis of bacterial genome variation at the population 115 fied in the two outbreaks occurred in Tuscany in 2012 and level. BMC Bioinformatics 2010; 11: 595). 116 2015. The same sublineage 11.2 was also responsible of out- The authors thanks F. P. D'Ancona for the collaboration 117 breaks among MSM in France and UK. It seems that the menin- in the National Surveillance System of Invasive Bacterial 118 gococci of cc11/ET15 are rather homogeneous, but different Diseases and in particular for the data collection system for 119 strains may emerge in different regions of the world. meningococcal invasive disease; A. M. Bartolesi Microbi- 120

Before drawing conclusions, possible limits of the study ology and Virology Unit, Florence Careggi University Hospi- 121

should be mentioned. First of all, data on the carriage of tal for sending samples and N. meningitidis strains from 122

MenC strains involved in the outbreak are not available. To Florence Hospital, Careggi; M. Comanducci, for helpful 123

this regard, although several studies showed low rates of discussion. 124

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