Epidemiological and Molecular Characterization of Invasive Meningococcal Disease in Italy, 2008/09-2012/13

Background Following the introduction of meningococcal serogroup C conjugate vaccine in Italy in 2005, changes in the epidemiology of Invasive Meningococcal Disease (IMD) were expected. The study aims were to describe the epidemiological trend and to characterize the isolates collected during the period 2008/09-2012/13 by multilocus sequence typing (MLST). Data on laboratory confirmed meningococcal diseases from National Surveillance System of IMD were reported. Methods Poisson regression models were used to estimate the incidence rate over time. Serogrouping and MLST were performed following published methods. Results The incidence rate of laboratory confirmed meningococcal disease decreased from 0.33 per 100,000 population in 2008/09 to 0.25 per 100,000 population in 2012/13. The serogroup B incidence rate was significantly higher (p<0.01) than that of other serogroups, among all age groups. The significant decrease of the IMD incidence rate (p = 0.01) reflects the decrease of serogroup B and C, in particular among individuals aged 15–24 years old (p<0.01). On the other hand, serogroup Y incidence increased during the period (from 0.01/100,000 in 2008/09 to 0.02/100,000 in 2012/13, p = 0.05). Molecular characterizations revealed that ST–41/44 cc and ST–11 cc were the main clonal complexes identified among serogroup B and C isolates, respectively. In particular, ST–41/44 cc was predominant in all age groups, whereas ST–11 cc was not identified in infants less than 1 year of age. Conclusions IMD incidence declined in Italy and serogroup B caused most of the IMD cases, with infants having the highest risk of disease. Continued surveillance is needed to provide information concerning further changes in circulating meningococci with special regard to serogroup distribution. Moreover, knowledge of meningococcal genotypes is essential to detect hyper-invasive strains.


Results
The incidence rate of laboratory confirmed meningococcal disease decreased from 0.33 per 100,000 population in 2008/09 to 0.25 per 100,000 population in 2012/13. The serogroup B incidence rate was significantly higher (p<0.01) than that of other serogroups, among all age groups. The significant decrease of the IMD incidence rate (p = 0.01) reflects the decrease of serogroup B and C, in particular among individuals aged 15-24 years old (p<0.01). On the other hand, serogroup Y incidence increased during the period (from 0.01/100,000 in 2008/09 to 0.02/100,000 in 2012/13, p = 0.05). Molecular characterizations revealed that ST-41/44 cc and ST-11 cc were the main clonal complexes identified among serogroup B and C isolates, respectively. In particular, ST-41/44 cc was predominant in all age groups, whereas ST-11 cc was not identified in infants less than 1 year of age.
Conclusions IMD incidence declined in Italy and serogroup B caused most of the IMD cases, with infants having the highest risk of disease. Continued surveillance is needed to provide information Introduction Invasive Meningococcal Disease (IMD) remains an important public health concern worldwide, for its global epidemiology and the burden of IMD in different countries [1,2]. IMD may be severe, often disabling, and sometimes fatal, especially among children, [3]. Although IMD occurs sporadically and outbreaks are rare, in some regions of the "African meningitis belt" devastating epidemic waves have been reported, [4].
The majority of Neisseria meningitidis strains which cause the invasive disease in Europe belongs mainly to serogroups B and C [5]. Of note, the groups at risk for IMD are patients less than 5 years of age, adolescents and young adults, [6].
Following the implementation of the meningococcal serogroup C conjugate (MCC) vaccination in many European countries, a decline in meningococcal serogroup C disease incidence has been observed, even if this serogroup is still circulating and is even responsible of small outbreaks, [7,8,9,10].
The MCC vaccine in Italy, introduced in 2005, is currently recommended in the National Immunization Plan (NIP 2012-2014), following regional policies, to all children from the 13 th to the 15 th month of age, and to 11-18 years old individuals, if not previously vaccinated, [11]. Data relating to MCC vaccine coverage was already described and estimated around 36.9% at the 12 th -24 th months for the cohort 2006 and around 72% at 24 th months for the cohort 2009, [12,13].
In Italy, in a previous analysis IMD due to serogroup C meningococci affected mostly children less than 4 years of age and adolescents, [14], but now the serogroup B is highly predominant, [15].
With regard to the licensure of the multicomponent vaccine against serogroup B vaccine (Bexsero 1 ) in Europe [16], it is recommended following regional immunization strategies throughout the country.
Since several reports evidenced the antigenic and genetic diversity among N. meningitidis isolates, it is important to identify the molecular characteristics of the circulating meningococci [17].
In Italy, the National Surveillance System of IMD, as part of the National Surveillance System for Invasive Bacterial Diseases, and its European networking (European Centre for Disease Prevention and Control, ECDC), is a long-standing surveillance through the country.
In this study, we used the information derived from the National Surveillance System with the following aims: a) to describe IMD cases occurring in Italy from 2008/09 to 2012/13; b) to describe the clonal complexes of the circulating disease-associated meningococci.

Data sources
In Italy, all cases of IMD is based on mandatory notification. Information regarding age, sex, clinical picture, vaccination status, outcome of the disease, municipality of residence, nationality, municipality where the case occurred is routinely collected and managed using a dedicated database. Clinical isolates or samples are collected and stored at -80°C at the National Reference Laboratory (NRL) of the Istituto Superiore di Sanità (ISS).

Bacterial strains and serogrouping
Meningococci are cultured on Thayer Martin agar plate with IsoVitalex 2% (Oxoid, Italy) and incubated in 5% CO 2 atmosphere at 37°C for 18h. Serogroup identification, performed at local level, is confirmed by the NRL; moreover, the NRL performs serogroup identification when needed. Serogroup identification is carried out by slide agglutination with commercial antisera (Remel Europe Ltd, UK) or by PCR testing [18].

DNA Extraction and MLST
Genomic DNA was extracted by QIAamp DNA minikit (Qiagen, Germany), according to the manufacturer's protocol for Gram-negative bacteria.
Detection of DNA of N. meningitidis from sterile body site (cerebrospinal fluid or blood) was performed by real-time TaqMan PCR assay commercial kit (BIOSENSE, Italy).
MLST was performed following the guidelines included in the Neisseria pubMLST website to identify sequence type and clonal complex (cc) (http://neisseria.org/neisseria/) [17].

Ethical Statement
Ethical approval for the study was not required, because all the examined cases were managed as part of routine diagnostics (standard care). Patient's data were recorded anonymously in separated files under the umbrella of the national surveillance system.
Poisson regression models were used to evaluate the temporal trend of the incidence rates (both overall, and stratified by regions with low and high percentage of UNK serogroup, and by age group).

Invasive Meningococcal Disease
Out of 794 laboratory confirmed cases of meningococcal disease in Italy, from 2008/09 to 2012/13 epidemiological years, 410 meningococcal isolates and 31 positive clinical samples were sent to the NRL.
The overall incidence of laboratory confirmed meningococcal disease during the period was 0.26 per 100,000 inhabitants ( Table 1).
The serogroup was unknown in 185 cases (185/794; 23%) out of the total (Table 1). For this reason, the analysis regarding the serogroups was performed on the overall country and on the Italian Regions divided in two groups: regions with <20% and regions with 20% of annual cases with UNK serogroup. A total of 491 IMD cases occurred in regions <20% of annual cases with UNK serogroup, and 303 cases in regions with a rate of annual cases with UNK serogroup 20% ( Table 1).
The total incidence of laboratory confirmed meningococcal disease significantly declined from 0.33 per 100,000 inhabitants in 2008/09 to 0.25 per 100,000 inhabitants in 2012/13 (p = 0.01), (S1 Fig). A similar trend was observed in regions with <20% of annual cases with UNK serogroup (p = 0.02) (S1 Fig). No significant changes in the incidence in the regions with 20% of annual cases with UNK serogroup was observed (p = 0.40), (data not shown).
Infants under 1 year of age (3.42/100,000) and children aged 1 to 4 years, (1.24/100,000) were the most affected. The lowest of the total IMD incidence rate was observed in adults 25 years (0.12/100,000), (Table 2).
Serogroup B incidence rate was significantly more common than all other serogroups, in all age groups (p<0.01) although a low difference in terms of incidence rate in the population aged 25 years was found. Serogroup C incidence rate decreased with the increase of the age. Serogroup Y incidence rate was higher among infants>1 year and children aged 5-9 years (0.07/100,000), Table 2.
A similar age distribution of IMD cases was observed in regions with <20% of annual cases with UNK serogroup, ( Table 2). Fig 1 shows the temporal trend of the incidence rate for all serogroups combined and for the serogroup B and C, separately. For the age group <1 year, no significant changes in the incidence rates of all serogroups combined as well as for the serogroup B and C, respectively were observed (Fig 1A). For the age group 1-4, there was a significant decline for all serogroups  combined; a similar decrease was observed both for serogroup B and C, respectively, but this decline was not statistically significant ( Fig 1B). Regarding the other three age groups (Fig 1C,  1D and 1E), only for age group 15-24 years (Fig 1D) a statistically significant decline for the overall incidence rate, as well as, for the serogroup B and C, respectively, was observed. A total of 23 isolates (23/344; 6.7%), belonging to sequence types not currently assigned to any clonal complex, were classified as unknown (UNK).  (Table 3).

Discussion
Invasive meningococcal disease (IMD) is a serious illness that despite an early intervention and modern intensive care can become fatal in few hours. Vaccination is the only strategy to prevent the disease and to improve the herd protection in the population, thus reducing morbidity and mortality. According to IMD surveillance data reported by ECDC, the incidence of confirmed cases in 28 European countries continues to decrease: from 0.95 per 100,000 in 2008 to 0.68 per 100,000 in 2012 [5].The main reason for this decline could be the introduction of MCC vaccination in several countries [20]. Moreover, the effect of vaccination on carriage status results in a long-term reduction of the disease [21].
In Italy, IMD incidence rate showed cyclical fluctuations, in particular regarding to the total number of cases due to serogroup B and C, respectively [22,14]. Moreover, other factors such as climate and geographical variation have been associated to a gradient in the IMD incidence in the country [23].
The risk of meningococcal disease clearly varies with age and serogroup. Meningococcal carriage is most common in teenagers whereas the invasive disease is reported mainly in infants and in a secondary peak observed in teenagers [24].
The serogroup distribution observed in this study was similar to that reported in most European countries, with serogroup B and C responsible for the majority of disease, followed by serogroup Y [5]. Serogroup B was responsible for the majority of IMD cases in all age groups, with a statistically significant higher incidence compared to any other serogroup. As already reported in Italy, serogroup B is the predominant among infants [15]. The Bexsero has been recently introduced in Italy even if with differences among the Regions. In a previous study [25], the potential Bexsero strain coverage has been estimated at 87% (95% CI 70-93).
In comparison to other serogroups, the number of IMD cases due to serogroups A and W is rare, in Italy. Whereas, an increase of IMD due to serogroup W have been reported in England and Wales since 2009 due to the emergence and clonal expansion of a single clone [26]. Serogroup Y increased as already reported in other European countries and worldwide [27,28,29]. These results may contribute in the revision of catch-up or booster vaccination, hopefully with the quadrivalent conjugate vaccine ACWY. The vaccine is available and recommended in Italy for at risk groups and for people who live or travel to countries where meningococcal disease is hyperendemic or epidemic. Moreover, it can be administered to children from 12 months of aged who have not received MCC vaccine or to adolescents aged between 12 and 16 years, as booster of MCC vaccine and for a complete coverage.
In the present analysis, serogroup B meningococci resulted more heterogeneous than serogroup C. In particular, the majority of clonal complexes among serogroup B were: ST-41/44 cc, ST-32 cc, ST-162 cc and ST-269 cc. These clonal complexes were distributed in all age groups.
The clonal complexes among serogroup C meningococci isolated from 2008/09 to 2012/13 were: ST-11 cc, ST-334 cc and ST-8 cc. Serogroup C ST-8 cc, mainly in persons aged less than 25 years, decreased and the serogroup C ST-334 cc, identified in adults, still increased. The majority of serogroup C ST-11 cc isolates were identified in all age groups except for infants less than 1 year of age, as already described [30].
Previously, serogroup C IMD cases in Italy were mainly associated with ST-8 cc (2003ST-8 cc ( -2005 [31] and ST-11cc (2007ST-11cc ( -2008 [32]. As with any national surveillance study, the estimation is likely to represent a possible underreporting of disease incidence. Moreover, the relatively high proportion of cases with UNK serogroup (23.3%) could affect the data. Nevertheless, results from Regions with a low rate of UNK serogroup in our dataset are consistent with those collected in the rest of the country.
In conclusion, data from the national surveillance system provides information on IMD in the country up to 2013, before the introduction of meningococcal B vaccination in the country. Molecular typing analysis permits to monitor the changes in the distribution of those hypervirulent clonal complexes recognized as responsible of outbreaks or rapid endemic expansion, demanding a continuous surveillance for the possible genetic pressure due to the immunization policies.