Carriage meningococcal isolates with capsule null locus dominate among high school students in a non-endemic period, Italy, 2012–2013

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Abstract

Meningococcal disease incidence in Italy remains quite low in the overall population except for infants. Within a study on carriage isolates among high school students we aimed to define: i) the prevalence of carriage isolates, ii) the phenotypic and iii) the molecular features of meningococci by Whole Genome Sequencing (WGS).

A total of 1697 pharyngeal samples from undergraduate students (age range 14–19 years) were collected from 2012 to 2013 from six larger cities in Italy. One hundred and twenty culture positive meningococci (7%) were analyzed. Carriage isolates were sent to the National Reference Laboratory for invasive meningococcal disease (IMD) for PCR-based serogroup identification, Multilocus Sequence Typing, PorA and FetA typing. Moreover, factor H binding protein (fHbp), Neisseria Heparin Binding Antigen (NHBA) and Neisserial adhesin A (NadA) were typed. Core genome MLST (cgMLST) was performed on a subsample of 75 carriage isolates.

Capsule null locus (cnl) predominated (47%), followed by serogroup B (27%). The antimicrobial susceptibility profile revealed an high prevalence of reduced susceptibility to penicillin G (54%) and a full susceptibility to ceftriaxone, ciprofloxacin and rifampicin. Carriage isolates presented a high genetic diversity: the clonal complexes (ccs) cc1136, cc198 and cc41/44, were the predominant. An high heterogeneity was also observed for PorA and FetA types. The fhbp and nhba genes were identified in all the carriage isolates; only 5% of the carriage isolates presented the nadA gene. The core genome MLST analysis revealed that the majority of the cnl isolates clustered in a distinct group.

The evidence gathered during this study provides the estimate of carriage isolates in high school students in a non-epidemic period in Italy that was lower than expected. Moreover, the highest proportion of carriage isolates were cnl and, overall, they were molecular heterogeneous.

Introduction

N. meningitidis is often asymptomatically carried at the mucosal surface of the nasopharynx and transmitted through respiratory secretions (Stephens et al., 2007; Soriano-Gabarró et al., 2011). Carriage status plays an important role in the dynamic of transmission of N. meningitidis (Soriano-Gabarró et al., 2011; Stefanelli and Rezza, 2016).

Many reports have been described that the microbiological traits of carriage isolates together with the age of the subject play a role for the carriage status (Christensen et al., 2010; Harrison et al., 2015). Most of the studies depicts an high incidence rate in 15 to 24 years old individuals (Christensen et al., 2010). In particular, Rønne et al. (1993) reported a prevalence of carriage from 19.8% to 20.4% in subjects between 16 and 20 years of age; moreover, Fraser et al. (1973) estimated a prevalence of carriage of 25.7% in a cohort of young people, 15 to 16 years of age.

In Italy, studies on the prevalence of meningococcal carriage isolates have been carried out in the years (Esposito et al., 2013; Gasparini et al., 2014; Germinario et al., 2010; Stroffolini et al., 1990; Tafuri et al., 2012). A study conducted in subjects 14–22 years of age demonstrated a meningococcal carriage peak of 25.5%, in the years 2011 and 2012 (Gasparini et al., 2014).

Few data are available on meningococcal carriage isolates in Europe, in fact, most of them refer to the local meningococcal epidemiology (Soriano-Gabarró et al., 2011).

Overall, the data reported in a number of carriage studies underline the indirect protection mediated by the immunization programs on carriage status as a key element in the dynamic of IMD (Claus et al., 2005; Maiden et al., 2008; Yazdankhah and Caugant, 2004).

In Italy, the quadrivalent meningococcal conjugate vaccination is currently recommended for adolescents aged 12–18 years (http://www.salute.gov.it/imgs/C_17_pubblicazioni_2571_allegato.pdf). Meningococcal B (MenB) and C (MenC) vaccinations are also recommended for primary vaccination (http://www.salute.gov.it/imgs/C_17_pubblicazioni_2571_allegato.pdf).

In 2012–2013, we conducted a carriage study among undergraduate students in six large cities in Italy in a non-epidemic period and prior to widespread availability of MenB vaccines in the country. Furthermore, carriage meningococcal isolates were characterized with the aim to: i) identify the carrier prevalence and the main serogroup of the carriage isolates; ii) determine their susceptibility against 4 antimicrobials; iii) identify, using Whole Genome Sequencing (WGS), or Sanger sequencing, the Sequence Types (ST), the clonal complex (cc), and the antigens comprised in the meningococcal B vaccines.

Section snippets

Ethical approval

The study protocol was accepted by the Ethics Committee of the Istituto Superiore di Sanità (reference number CE/11/313). A written informed consent form was obtained from parents of participants aged <18 years old. Participants aged 18 years or above gave their informed consent.

Study design and population

The collection was carried out from June 2012 to July 2013. A total of 1697 healthy teenagers aged between 14–19 years, attending secondary schools in six different Italian cities, were invited voluntary to participate

Prevalence of meningococcal carriage isolates

From the 1697 teenagers enrolled, 120 resulted culture positive for N. meningitidis. In particular, the overall carriage rate was 7% (95% CI, 5.90–8.40), that varied among the collaborating centers: 4.0% in Napoli (12/300) and 4.2% in Padova (11/260), 5.3% (16/300) in Roma, 7.5% (17/225) in Bologna, 9.5% (31/315) in Milano and 11.5% (34/296) in Torino (Fig. 1). The median age of the carriers was 17.5 years (range 14–19 years). Fig. 2 shows the distribution of the carriers by age. The highest

Discussion

Although the rate of IMD in Italy is currently low, outbreak and small clusters have been recently reported (Miglietta et al., 2018; Stefanelli et al., 2018). Meningococcal carriage influences the dynamic of the spread of the disease in particular for specific age group (Caugant and Maiden, 2009).

We aimed to evaluate the carriage rate and the molecular characteristics of carriage isolates from high school students prior to the introduction of MenB vaccines in Italy and in a non-epidemic period (

Competing interests

No competing of interest.

Authors' contributions

AN,CF, PV, performed the microbiological analyses on samples. MPL, LR, LD, IS, collected the swabs at local level and identified Neisseria meningitidis. AC, AA, FL performed the whole genome sequencing on a subsamples of meningococcal isolates. CF, PV, AN contributed in the analysis of carriage isolates and together with PS revised critically the manuscript. PS conceived the study and together with AN wrote the manuscript. All authors have read and accepted the submitted manuscript.

Acknowledgements

The authors are grateful to all students and parents who participated in the study reported in this article.

The authors thanks Antonella Fortunato, Anna Maria Palozzi and Silvia Fillo (Scientific Department, Army Medical Center, Rome, Italy) for the whole genome sequencing; Annapina Palmieri (Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy) for the collaboration in the National Surveillance System of Invasive Bacterial Diseases and for the data collection; Fenicia

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