Phenotypic characterization of Neisseria meningitidis strains isolated from invasive meningococcal disease in Brazil from 2002 to 2017

Introduction Invasive meningococcal disease (IMD) has a high rate of fatality and may cause severe clinical sequelae. Over the years, the epidemiology of IMD has changed significantly in various regions of the world, and laboratory surveillance of this disease is important for mapping epidemiologic changes. Aim To perform phenotypic characterization of Neisseria meningitidis strains isolated from invasive disease in Brazil from 2002 to 2017, as a complementation of the data obtained in the period of 1990–2001. Methodology In total, 8,689 isolates sent to Adolfo Lutz Institute confirmed as N. meningitidis by conventional methods were serogrouped by slide agglutination against MenA, MenB, MenC, MenE, MenW, MenX, MenY and MenZ; serotyped and serosubtyped by a whole-cell dot-blotting assay with monoclonal antibodies. Results The isolates were sent from all regions of Brazil, and the southeast region was responsible for the largest number of isolates (57.2 %). Overall, the total sample (n=8,689) was represented by serogroups C (n=4,729; 54.4 %), B (n=3,313; 38.1 %), W (n=423; 4.9 %), Y (n=203; 2.3 %), X (n=5; 0.1 %) and others (n=16; 0.2 %). A shift in the prevalence of serogroups was observed in 2006, when serogroup C became the most prevalent (65.5 %), surpassing the serogroup B (21.9 %). The main isolated phenotypes were C:23:P1.14–6; B:4,7:P1.19,15; W:2a:P1.5 and W:2a:P1.5,2. Conclusion The data show an important change in the distribution of meningococcal serogroups, serotypes and subtypes occurring during 2002–2017. A continuous laboratory-based surveillance provides robust information to implement appropriate strategies to IMD control.


InTRoduCTIon
Invasive meningococcal disease (IMD) is one of the most feared diseases due to its rate of fatality of 5-10 %, and causes severe clinical sequelae such as deafness, amputation and mental impairment [1]. Twelve serogroups of Neisseria meningitidis have been characterized based on their capsular polysaccharides, although serogroups A, B, C, W, Y and X are the most clinically significant [2,3]. Most cases of IMD occur sporadically and vary according to the age, socioeconomic conditions, geographic regions and serogroups involved; outbreaks and epidemics may occur at irregular intervals [4][5][6]. Since 1975, IMD is a notifiable disease in Brazil and the National Surveillance System (SNV) monitors the epidemiological characteristics of the disease.
From 1990 to 2001, 68, 332 IMD cases were reported to the Brazilian National Notifiable Disease Surveillance System (SINAN), and the annual incidence rate was estimated to be around 1-3 cases per 100, 000 persons [7]; in contrast, in the period from 2002 to 2017, 41,192 IMD cases were notified and the annual incidence rate was estimated at around 0.55-2.12 cases per 100,000 persons, presenting a clear reduction trend in the period, from 2.12 cases in 2002 to 0.55 cases per 100, 000 in 2017 [8]. Thirty-one percent (n=12, 621) of the cases notified between 2002 and 2017 were confirmed by isolation of the causative strain, from which 68.8 % (n=8,689) were referred to the National Reference Laboratory for Meningitis at Adolfo Lutz Institute, São Paulo, herein named IAL, for species confirmation and further characterization.
Over the years, the epidemiology of IMD has changed significantly in various regions of the world. The aim of this study was to analyse the phenotypic characteristics of N. meningitidis strains causing IMD in Brazil, thus complementing the previously described results [7] of the period from 1990 to 2001.

Meningococcal isolate collection
The IAL receives invasive meningococcal isolates from public health laboratories and hospitals from all Brazilian regions for full phenotypic characterization.
Of the 12, 621 cases of IMD laboratory-confirmed by culture between 2002 and 2017 in Brazil, 8,689 isolates (68.8 %) were sent to IAL.

Identification and serogrouping of N. meningitidis
All the 8,689 IMD isolates were confirmed as N. meningitidis by conventional methods (oxidase test and carbohydrate utilization testing for glucose, maltose, sucrose and lactose). Serogrouping was performed by slide agglutination with polyclonal goat or horse antisera, prepared at the IAL against MenA, MenB, MenC, MenE, MenW, MenX, MenY and MenZ, as described previously [9,10].
The distribution of isolates by serogroup is shown in Fig. 1 [7,12]. However, from 2002 the incidence of serogroup B began to decline, and, in 2006, it was surpassed by a new emerging meningococcal serogroup C clone, C:23:P1.14-6. This serogroup C belonging to ST-103 CC, was described for the first time associated with an epidemic situation in Brazil [13].  [14]. The ST-103 CC has been also described among sporadic cases of IMD by serogroup C in the Czech Republic accounting for less than 1 % of serogroup C isolates [15].  Table 1). This new MenC clone remains currently the main cause of IMD in Brazil, although with a lower incidence rate of 0.17 per 100,000 in 2017, due to the introduction in 2010 of a Meningococcal C Conjugate Vaccine (MCC vaccine) into the national immunization program. Initially, the strategy was to vaccinate children at 3 and 5 months, with a booster dose at 12 months of age, and no catch-up campaigns. After implementation of this vaccine, the IMD overall rate has decreased from 1.54 per 100,000 in 2010 to 0.55 per 100,000 in 2017 [8]. As shown in Table 1, there has been a substantial reduction in the frequency of serogroup C isolates from infants and older children, but the prevalence of serogroup C isolates remained relatively high in adolescents and young adults. As suggested by Andrade et al. [21], the reduction of IMD cases in children was mostly due  to the direct effect of the vaccination. To provide protection for the older groups, serogroup C vaccination for adolescents aged 12 to 13 years was implemented in March 2017, and by 2020 the age range will be gradually increased, starting at 9 years of age [21]; http://www. aids. gov. br/ pt-br/ legislacao/ nota-informativa-no-3112 016c gpni devi tsvsms.
Although serogroup C remains prevalent in all regions of Brazil, the increased incidence of serogroup W in the Southern states from 2014 is of concern because of its potential to cause epidemics, severe disease and its high case fatality rate as has been seen in other countries [22,23] (    Serogroup Y has been most commonly related to carriage isolates over time [27,28] but this situation has changed in many countries like the USA, for example, where it has become (during the latter half of the 1990s) an important cause of IMD. The increase in the prevalence of serogroup Y in the USA is attributed to the emergence of a ST-23 clone and, while the incidence of all serogroups has fallen in the USA in recent years, serogroup Y continues to be responsible for approximately a third of endemic disease cases [29]. The proportion of MenY disease has also increased during the last years in some European countries such as Sweden, Finland, England and Wales, which may be in part due to the use of MenC conjugate vaccine as opposed to the quadrivalent serogroup ACWY vaccine [3]. The most frequent genotype that has been described is Y:P1.5-2,10-1 CC-23 [30,31]. As presented in Tables 2 and 3, serogroup X showed a single phenotype, 4,7:P1.19,15 (5/5; 100 %).

Conclusion
The data presented herein show an important change in the IMD epidemiological scenario due to a significant shift from serogroup B to serogroup C observed from 2006 onwards, when serogroup C became the most frequent capsular type.
Performing a continuous laboratory-based surveillance highlighting the changes over time in serogroups and serotypes:serosubtypes distribution of invasive N. meningitidis contributes to the knowledge of the IMD epidemiology in Brazil, providing robust information to implement appropriate strategies to the IMD control.

Funding information
The authors received no specific grant from any funding agency.