Molecular characteristics of Neisseria meningitidis carriage strains isolated from Korean adolescents

Han Wool Kim, MD, PhD 1; Soyoung Lee, MD,PhD 1; Daeho Kwon, PhD2; Seo Hee Yoon, MD, PhD3; Kyung-Hyo Kim, MD, PhD 1,4

Center for Vaccine Evaluation and Study, Ewha Womans University School of Medicine 1; Department of Microbiology, Catholic Kwandong University, College of Medicine2;

Department of Pediatrics, Severance Hospital, Yonsei University College of Medicine3; Department of Pediatrics, Ewha Womans University School of Medicine 4, Seoul, South Korea

Poster 57118

ABSTRACT

In this study, we provided the first report on the carriage rate, serogroup,and MLST of menincococcal carrier isolates from Korean adolescents. Theobserved carriage rate was lower than other country adolescents. Like otherWestern countries, meningococcal serogroup B has emerged in Korea, andhypervirulent clones such as CC-269 were identified. Further monitoring of N.meningitidis and assessing the potential strain coverage of meningococcalvaccines, in particular meningococcal B vaccine, is necessary.

Laboratory methodBacterial identification• The oropharyngeal swabs were immediately placed in

transport media (Culture Swab™ Plus Amies Gel with Charcoal; BD Diagnostic Systems, Sparks, MD) stored in room temperature and transport within four hours to Ewha Center for Vaccine Evaluation and Study, Medical Research Institute, Ewha Womans University School of Medicine.

• Swabs were plated onto selective medium (Thayer-Martin) and incubated in 5% CO2 at 37°C

• Morphological evaluation recognized as possible

colonies (round, smooth, moist, glistening, convex and gray) of N. meningitidis were plated on chocolate agar

• MALDI-TOF (VITEK-MS, BioMerieux, France) (Figure 2): All of the identifications at the genus or species levels with a score 90% were considered to be reliable identifications.

• Conventional method: oxidase, catalase positive, gram negative diplococci, sugar fermentation test

• crgA PCR: to confirm N. meningitidis species identify

Serogrouping by PCR • Genomic DNAs were purified using DNA purification kit (Promega, Madisonm, WI, USA)• PCR was performed for all meningococcal isolates by

using species-specific primers. (17)• Target gene - orf-2 (serogroup A),

siaD (Serogroup B, C, W135, Y)

Figure 3. Flow chart for laboratory methods

Figure 2. Scheme of MALDI-TOF

Multilocus Sequence typing by PCR (18)• Target gene – 7 housekeeping gene

abcZ, adk, aroE, fumC, gdh, pdhC, pgm• After amplification by PCR, sequencing of PCR

amplified fragments using the Sanger method. • The sequences are compared with existing alleles on

the Neisseria MLST website (http://pubmlst.org/ neisseria/) for determination of allele numbers, sequence types (STs), and clonal complexes (CC) of the isolate. New alleles and STs were submitted to the MLST website for assignment.

• eBURST v3 software was used to estimate the relationships among the isolates

IntroductionNeisseria meningitidis is a diverse commensal bacterium thatoccasionally causes severe invasive diseases. Therelationship between carriage and invasive disease is notfully understood. The analysis of isolates based bymultilocus sequence typing (MLST) is improving theunderstanding of the dynamics of meningococcaltransmission. The meningococcal sequence type wasinvestigated in the meningococcal carriage strains isolatedfrom adolescents in Korea.

MethodsFrom April 2015 to May 2015, the oropharyngeal sampleswere collected from the sophomore year of high schools inKorea. Neisseria meningitidis were identified using Vitek MSsystem (bioMérieux, France) and all isolates werecharacterized by molecular serogrouping and MLST.

ResultsOf the 1,460 oropharyngeal swabs, 56 isolates (carriagerate, 3.4%) were identified as Neisseria meningitidis.Serogroup B was the most common serogroup, followed byserogroup C, then by serogroup Y. By MLST analysis, 26different sequence types (ST) were identified in this study.The most frequent ST was ST-3091 (9 strains), ST-11278(7), and ST-44 (5). They belonged to clonal complex (CC)-269, CC-32, and CC-41/44, known as hyper-virulent clones.The distribution of serogroups and ST is illustrated in theeBURST diagram.ConclusionLike other Western countries, meningococcal serogroup Bhas emerged in Korea, and hypervirulent clones such as CC-269 were identified. Further monitoring of N. meningitidisand assessing the potential strain coverage ofmeningococcal vaccines, in particular meningococcal Bvaccine, is necessary.

BACKGROUND

Study population and recruitment

- Cross sectional, descriptive studyconducted from April to May, 2015

- Study population: 9 high schools located in Gyunggi, Korea

- Inclusion criteriaFirst grade studentsAgree to participate in the study by signing written informedconsent from the subjects and their parents

- Exclusion criteriaCranio-facial malformations prohibiting the collection ofposterior pharyngeal swab

Oropharyngeal swab: A trained physician took a posterior oropharyngeal swab (CultureSwab™ Plus Amies Gel with Charcoal; BD Diagnostic Systems,Sparks, MD) from behind the uvula, the posterior wall of theoropharynx.

Figure 1. Map of the study region

MATERIALS AND METHODS

Neisseria meningitidis is a globally important causative agent of sepsis and meningitis with substantial morbidity andmortality (1). Even with appropriate treatment, the case fatality rates ranged from 5% to 15% (2). The pharyngeal carriageof N. meningitidis is a prerequisite for invasive meningococcal disease (3). This bacterium can cross the epithelial barrier ofthe pharynx to enter the bloodstream causing septicemia. Subsequently, it can cross the blood-brain barrier giving rise tomeningitis (4). However, meningococcal carriage is in most cases asymptomatic and results in strain-specific immunity (5).Carrier status can be regarded as a natural booster that contributes to the spontaneous acquisition of herd immunity (6).Meningococcal carriage rates are variable by age, with peak prevalence carriage rates usually ranging from 10% to 35% inadolescents and young adults (7, 8). Male sex (9-11), cigarette smoking (12, 13), passive exposure to smoke (13), kissing(7, 11), and overcrowding (7) are associated with carriage, and many of these factors are also risk factor for meningococcaldisease (14, 15).To understand epidemiology and pathogenesis of meningococcal disease, the carriage state needs to be investigated.Although many carriage studies on various populations were reported in Europe and United States (8), only incompleteinformation is available in Korea. Because of the high mortality and prevalence of meningococcal infection in Korean army,the epidemiology of meningococcus has been mostly evaluated in the army. In spite of high risk for meningococcal diseasesin adolescents, only two cases studies have been reported. Choi et al. (16) reported the carriage rate in high schoolstudents. However, this result was associated with meningococcal meningitis case and study population was too small (N =78). Moreover, no serological or genetic characterization of isolates were evaluated in any studies for adolescents.The aim of this study is to gain information concerning the carriage of N.meningitidis among Korean adolescents. In thisreport, we provided the first report on the carriage rate, serogroup, and multilocus sequence typing (MLST) ofmeningococcal carrier isolates from the first grade students of high school in Korea.

MATERIALS AND METHODS

CONCLUSION REFERENCES

1. WHO practical guidelines. 1998.2. Howitz M, et al. Morbidity, mortality and spatial distribution of meningococcal disease, 1974-2007. Epidemiol Infect. 2009;137(11):1631-40.3. Gasparini R, , et al. Neisseria meningitidis, pathogenetic mechanisms to overcome the human immune defences. J Prev Med Hyg. 2012;53(2):50-5.4. Caugant DA. Genetics and evolution of Neisseria meningitidis: importance for the epidemiology of meningococcal disease. Infect Genet Evol. 2008;8(5):558-65.5. Goldschneider I, , et al. Human immunity to the meningococcus. II. Development of natural immunity. J Exp Med. 1969;129(6):1327-48.6. Guzzetta G, , et al. On the relationship between meningococcal transmission dynamics and disease: remarks on humoral immunity. Vaccine. 2009;27(25-26):3429-34.7. MacLennan J, , et al. Social behavior and meningococcal carriage in British teenagers. Emerg

Infect Dis. 2006;12(6):950-7.8. Christensen H, , et al. Meningococcal carriage by age: a systematic review and meta-analysis. Lancet Infect Dis. 2010;10(12):853-61.9. Cartwright KA, , et al. The Stonehouse survey: nasopharyngeal carriage of meningococci and Neisseria lactamica. Epidemiol Infect. 1987;99(3):591-601.10. Caugant DA, et al. Asymptomatic carriage of Neisseria meningitidis in a randomly sampled population. J Clin Microbiol. 1994;32(2):323-30.11. Neal KR, et al. Changing carriage rate of Neisseria meningitidis among university students during the first week of term: cross sectional study. BMJ. 2000;320(7238):846-9.12. Stuart JM, , et al. Effect of smoking on meningococcal carriage. Lancet. 1989;2(8665):723-5.13. Riordan T, et al. Acquisition and carriage of meningococci in marine commando recruits. Epidemiol Infect. 1998;121(3):495-505.

14. Baker M, et al. Household crowding a major risk factor for epidemic meningococcal disease in Auckland children. Pediatr Infect Dis J. 2000;19(10):983-90.15. Stanwell-Smith RE, , et al. Smoking, the environment and meningococcal disease: a case control study. Epidemiol Infect. 1994;112(2):315-28.16. Choi S, et al. Clinical and Epidemiologic Features of Meningococcal Infections in Incheon, Korea. Infection and Chemotherapy. 2005;37(3):119-26.17. Taha MK. Simultaneous approach for nonculture PCR-based identification and serogroup prediction of Neisseria meningitidis. J Clin Microbiol. 2000;38(2):855-7.18. Birtles A, et al. Multilocus sequence typing of Neisseria meningitidis directly from clinical samples and application of the method to the investigation of meningococcal disease case clusters. J Clin Microbiol. 2005;43(12):6007-14

Oropharyngeal swab

from the posterior wall of oropharynx behind

uvula

Inoculation on selective agar

Thayer-Martin chocolate agar

Bacteria collection and selection

Transport medium

Transwab ® Stuart’s charcoal

Selection of colonies

Round, smooth, moist, glistening and convex

Acceptable: confidence of result >90%MALDI-

TOF

(Vitek-MS, BioMerieux,

France)

Identification

Conventional method

: oxidase, catalase positive, gram negative diplococci, sugar fermentation test (glucose and maltose positive)

Not acceptable:

Discrepancy between 2 spots or confidence of any spot <90%

- Confirm N. meningitidis species: crgA

-Serogrouping: A (orf-2),

B,C,W135,Y (siaD)

- MLST: 7 housekeeping gene

abcZ, adk, aroE, fumC, gdh, pdhC, pgm

- eBURST analysis

Molecular workup

RESULTS

Baseline characteristicsA total of 2,908 students in nine high schools were invited to participate to the study, 1,448 of whom declined (response rate, 50.2% [95% CI, 48.4 to 52.0%]). The reasons for refusal and their demographic data were not further investigated. Overall, 1,460 subjects were enrolled. The demographic characteristics of subjects are shown in Table 1.

Carriage rateNeisseria meningitidis was identified from 49 (3.4%) of 1,460 swabs (Table 1). Two or more meningococcal strains were identified from same swab in 6 subjects therefore a total of 56 strains was isolated. The carriage rate of each school was ranged from 1.3% to 6.8% and the highest rate of carriage was observed in school D (Table 1).

SerogroupsOf the 56 meningococcal isolates, 42.9% (24/56) of the isolates were nongroupable (Figure 4). Serogroup B was most frequent (15 isolates from 12 subjects, 26.8%). All isolates of serogroup Y were collected from school D (Figure 5).

Sequence types of carrier strains By MLST analysis, 26 different STs were identified in this study (Table 2). A total of 16 STs (ST-11937, 11938, 11939, 11940, 11941, 11942, 11943, 11946, 11947, 11948, 11949, 11950, 11951, 11953, 11954, and 11955) was new STs. The most frequent STs were ST-3091 (CC-269) and ST-11278 (CC-32). The distribution of serogroups and STs is illustrated in the eBURST diagram (Figure 6).

Table 1. Demographic data of subjects and carriage rate.

We thank Sanghyun Shin for enrollmentof participants. This study was fundedby GlaxoSmithKline (GSK). GSK weregiven the opportunity to review theposter for medical accuracy andprotection of intellectual property,however editorial control resided fullywith the authors only.

Acknowledgement

SchoolStudy month

No. of subjects

Male (N,%)Mean age

(year)No. of isolates

Carriage rate (N/%)

A April 243 116 (47.7) 15.1 9 7/2.9

B April 172 92 (53.5) 15.1 12 8/4.7

C April/May 176/77 253 (100.0) 15.2 10 10/4.0

D May 176 103 (58.5) 15.2 13 12/6.8

E May 80 45 (56.3) 15.1 3 3/3.8

F May 229 121 (52.8) 15.2 3 3/1.3

G May 55 38 (69.1) 15.1 2 2/3.6

H May 59 36 (61.0) 15.1 1 1/1.7

I May 193 98 (50.8) 15.3 3 3/1.6

Total 1460 902 (66.4) 15.3 56 49/3.4*

* 2 or more colonies were collected from 6 subjects

0

2

4

6

8

A B C D E F G H I

Num

ber

of

isola

tes

School

Serogroup B

Serogroup C

Serogroup Y

Nongroupable

15, 27%

12, 21%5, 9%

24, 43%

Serogroup B

Serogroup C

Serogroup Y

Non-groupable

Figure 5. Serogroup distribution of meningococcal isolates.

Table 2. Sequence types of isolates

Figure 6. eBURST analysis of 56 strains. The circle sizes correlate with the number of strains of each sequence type

Figure 4. Serogroup distribution of meningococcal isolates.

SerogroupNo. of isolates (% of isolates)

Sequence types (No. of isolates)

A 0 (0.0) -

B 15 (26.8)3091 (7), 44 (2),

35, 437, 5542, 11938, 11949, 11950 (1),

C 12 (21.4) 11278 (7), 32 (3), 5939, 11943 (1)

W-135 0 (0.0) -

Y 5 (8.9) 23 (4), 8579 (1)

Non-groupable

24 (42.9)44 (3), 11942 (3), 3091, 11937, 11940 (2)

437, 1136, 5542, 11939, 11941, 11946, 11947, 11948, 11951, 11953, 11954, 11955 (1)

Total 56