Longitudinal study of meningococcal carriage rates in university entrants living in a dormitory in South Korea

University students, especially those living in dormitories, are known to have a high risk of invasive meningococcal disease. We performed a longitudinal study to investigate the change in Neisseria meningitidis carriage rates and identify the risk factors for carriage acquisition in university students in South Korea. We recruited university entrants who were admitted to a student dormitory. Pharyngeal swabs were taken from participants at baseline, 1 month, and 3 months, and the subjects completed a questionnaire. Culture and real-time polymerase chain reaction (PCR) for species-specific ctrA and sodC genes were performed. The cultured isolates or PCR-positive samples were further evaluated for epidemiologic characterization using serogrouping, PorA typing, FetA typing, and multilocus sequence typing (MLST). At the first visit, we enrolled 332 participants who were predominantly male (64.2%) with a median age of 19 years. Meningococcal carriage rates increased from 2.7% (95% confidence interval [CI] 0.9–4.4%) at baseline to 6.3% (95% CI 3.4–9.0%) at 1 month and 11.8% (95% CI 7.8–15.6%) at 3 months. Nongroupable isolates accounted for 50.0% of all isolates, with serogroup B being the next most prevalent (24.1%). In the study population, male sex (OR 2.613, 95% CI 1.145–5.961, p = 0.022) and frequent pub or club visits (OR 3.701, 95% CI 1.536–8.919, p = 0.004) were significantly associated with meningococcal carriage. Based on serotype and MLST analyses, six carriers transmitted meningococci to other study participants. N. meningitidis carriage rates among new university entrants who lived in a dormitory significantly increased within the first 3 months of dormitory stay, probably owing to the transmission of identical genotype among students. Based on the risk of meningococcal disease, meningococcal vaccination should be considered for students before dormitory admission.


Introduction
Despite appropriate treatment, Neisseria meningitidis can cause life-threatening invasive meningococcal disease (IMD), including meningitis and/or sepsis [1]. The human pharynx is PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0244716 January 28, 2021 1 / 12 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a unique reservoir of N. meningitidis, and meningococci spread between close contacts via aerosol droplets released during coughing [2]. N. meningitidis infection often results in asymptomatic colonization of the human nasopharynx. Moreover, meningococcal transmission is easier through close contact with a N. meningitidis carrier than by close contact with an infected individual with IMD, underlining the importance of asymptomatic carriers [3,4]. While the overall carriage prevalence is estimated at 10%, the incidence rate of IMD varies between 0.5/100,000 in North America and up to 1,000/100,000 in epidemic settings [5,6]. In industrialized countries, carriage rates increase gradually through childhood (4.5%), peak during adolescence and young adulthood (23.7%), and stabilize during adulthood (7.8%). In developing countries, however, carriage rates generally peak in early childhood [7]. The carriage rate is also influenced by social risk factors, including going to clubs, kissing, smoking, and living in closed or semi-closed environments such as university dormitories [8]. In South Korea, only limited data are available. Moreover, the IMD annual incidence is low (0.01-0.08/ 100,000) and it occurs sporadically without clusters, other than the outbreak of army basic training center [1,9]. Although the Korea Centers for Disease Control and Prevention (KCDC) guidelines recommend vaccination for new dormitory entrants, little is known about vaccination and no educational institution requires it [10]. Given that few studies have analyzed the meningococcal carriage rates in South Korean young adults, especially students residing in dormitories, it is necessary to conduct an appropriate epidemiologic analysis. Considering the high mortality rate of the IMD, the recent increase in antibiotic resistance is also a problem [9,11].
This study aimed to assess the sequential changes in N. meningitidis carriage rates over a 3-month period in students newly admitted to a university dormitory in South Korea. In addition, we investigated the dominant circulating serogroups, antimicrobial susceptibility, and factors related to meningococcal pharyngeal carriage.

Study population
This prospective observational study was conducted during the first semester (March-June 2018) of the year at a university dormitory in Incheon, South Korea. We included first-year students, who resided in the same dormitory and provided their informed consent. An advertising email was sent to all students, and desks were placed at the entrance of the dormitory with a consent form to recruit the applicants. We excluded students who had a history of meningococcal vaccination, completed military service (quadrivalent meningococcal conjugate vaccine [MCV4] vaccination has been mandatory in the South Korean army since 2012), had taken antibiotics 2 weeks prior to the study, had taken immunosuppressants or steroid drugs 2 weeks prior to the study, or had existing immunological or hematological disorder. These exclusion criteria did not apply to re-visiting applicants.
We obtained approval from the Institutional Review Board of Severance Hospital (4-2017-1095), and collected written informed consent from all participants.

Collected data and specimens
Three visits were programmed for each participant; on dormitory admission, at 1 month, and at 3 months (end of the first semester). Participants completed the questionnaires during each visit, wherein the following data were collected: demographic characteristics; medical history including current medications; recent symptoms of upper respiratory tract infection (within the previous week); recent record of traveling abroad (within past 4 weeks); the number of club/pub visits (within past 2 weeks); smoking (active or not); alcohol consumption (frequency); sharing of cups and cigarettes (past 1 week); and the number of kisses (within past 4 weeks). Two oropharyngeal swabs, from the tonsils/the tonsillar fossa and the posterior pharynx, were collected simultaneously at each visit using BD CultureSwab MaxV Liquid Amies (Becton-Dickinson, USA) for conventional culture and molecular screening tests.

Microbiological tests
One swab was inoculated into modified Thayer-Martin media and stored for future analysis. The second swab was inoculated into Mueller-Hinton broth containing vancomycin (3 mg/L), colistin (7.5 mg/L), nystatin (1,250 U/L), and trimethoprim (5 mg/L) for enrichment. The broth was then incubated overnight, part of it inoculated into modified Thayer-Martin media, and the rest stored for molecular screening. The inoculated Thayer-Martin media from the direct swab and enriched broth were both incubated for 72 hours to allow for N. meningitidis growth. Suspected colonies were identified at 24, 48, and 72 hours with MALDI-TOF mass spectrophotometry (Bruker Daltonics). All isolates were stored in a deep freezer at -70˚C for further analysis. The stored enriched broth was tested for N. meningitidis-specific ctrA and sodC genes using real-time polymerase chain reaction (PCR) as previously described [12,13]. We undertook epidemiologic characterization of N. meningitidis isolates or PCR-positive swabs using the previously described molecular methods, such as PCR-based serogrouping, PorA typing, FetA typing, and multilocus sequence typing (MLST) [9]. Antimicrobial susceptibility testing was performed using Etest strips (bioMérieux), and the minimum inhibitory concentration (MIC) was interpreted using the Clinical Laboratory Standards and Institute (CLSI) guideline [14]. The processing of specimens in this study is shown in

Statistical analysis
This study was a descriptive study on N. meningitidis carriage. N. meningitidis carriers were defined as participants whose pharyngeal swabs showed an N. meningitidis-positive culture or real-time PCR. Continuous variables are presented as mean±standard deviation and categorical variables as numbers and percentages. Differences in proportion were tested with Fisher's exact test. Factors associated with the carriage and acquisition of N. meningitidis were evaluated using univariate and multivariate analyses. Covariates of the multivariate logistic regression model included all significant risk factors in univariate analyses and other well-known risk factors. The results of univariate and multivariate analyses for identifying risk factors are reported as adjusted odds ratios (ORs) and 95% confidence intervals (CIs). Statistical tests were two-sided, and a p-value <0.05 was considered statistically significant. Data were analyzed using SPSS statistics 23 (IBM, Armonk, NY, USA).

Risk factor analysis
Risk factor analysis of participant characteristics on N. meningitidis carriage in the study population is shown in Table 2. Univariate analysis showed no significant association between N. meningitidis carriage and current smoking, alcohol consumption, three or more roommates, international travel, recent antibiotics intake, recent upper respiratory tract infection, cup/cigarette sharing, and intimate contact. However, in multivariate analysis, male sex (OR 2.61, 95% CI 1.15-5.96) and recent pub or club visits (OR 3.70, 95% CI 1.54-8.92) were associated with N. meningitidis carriage during the study period. Factors associated with the acquisition of N. meningitidis for the 287 noncarriers at baseline were also significant for male sex (OR 3.58, 95% CI 1.34-9.57) and recent pub or club visits (OR 3.33, 95% CI 1.39-7.94), but they were not significant for a dormitory building (p = 0.344). The antimicrobial susceptibility of the cultured isolates is shown in Table 4. Of the 58 isolates, 42 were culture-positive, and the susceptibility test was performed on 41 available isolates, of which 26 had low penicillin susceptibility and seven were penicillin-resistant. Of the 26 isolates with low penicillin susceptibility, nine, five, and four were identified as serogroups B, C, and H, respectively, while eight were nongroupable.

Distribution of MLST sequence type by FetA and PorA typing
Of the 58 PCR-or culture-positive isolates, only 30 isolates were recovered from the specimens; MLST was performed on all 30 isolates. MLST of the 30 isolates showed that the most common sequence type (ST) was ST3091 (7/30; 23.3%), with all of these isolates belonging to clonal complex 269 and serogroup B (Table 5). Of the seven isolates with ST3091, five were F3-6 by FetA typing, while 19 and 15 subtypes by PorA typing. The second most common was clonal complex 178, consisting of ST178 and ST2397, all belonging to serogroup H, followed by clonal complex 32, consisting of ST11278 and one new sequence type. All clonal complex 32 isolates belonged to serogroup C and were not susceptible to penicillin and ampicillin. Interestingly, five isolates showed novel sequence types that were closely related to ST11940. All of the isolates in this clonal complex shared the same PorA1 types and were nongroupable.

Discussions
South Korea's national notifiable diseases surveillance system has reported a relatively low incidence of meningococcal disease, 0.01-0.08 cases per 100,000 persons. Nonetheless, previous reports on the seroprevalence and carriage rate of N. meningitidis in South Korean populations suggested a non-negligible burden of meningococcal disease in the country [1,9,[15][16][17]. N. meningitidis is a leading cause of bacterial meningitis in adolescents and young adults. Moreover, university students, especially those living in dormitories, are known to have an increased risk of meningococcal disease. This longitudinal study showed that N. meningitidis carriage rates significantly increased from 2.7% to 11.8% over the first semester among new university entrants in South Korea who were living in dormitories. In the isolates swabbed at the third visit, there were culture-negative, PCR-positive samples (n = 13), all of which were sodC-positive and ctrA-negative. The genotypes were not identified at the first and second In the case of multiple visits, the same isolate was identified in one participant.
Participants were classified according to the putative transmission events in Table 3, and the specimens of B1-1 and C1-1 could not be cultured. visits, and they were not included in the analysis as they were likely to be false-positive. However, the possibility of actual expansion of NG meningococci could not be completely ruled out, and the carriage rate increased to 16.7% (44/263) when the third visit included culturenegative, sodC-positive swabs. Previous studies of N. meningitidis carriage in South Korea have shown a carriage rate of 3.4% in healthy Korean adolescents, with serogroup B being the most common serogroup, followed by serogroups C, E, and Y [15]. In contrast, another study conducted on first-year university students in South Korea in 2009 did not show a significant increase in carriage rates in a different university dormitory [16]. Such contrasting observations might have resulted from the longer time interval between visits and the larger sample size in our study. More studies should be performed to clarify the transmission dynamics of N. meningitidis among university students in South Korea.
In the present study, nongroupable isolates were the most prevalent, followed by serogroup B. Similarly, Lee et al. evaluated the serogroup changes of N. meningitidis in South Korea between 2010 and 2016, and showed that the most common serogroup was serogroup B, which was isolated every year except for 2011. Moreover, Kim et al. found that serogroup B was the most common serogroup in Korean adolescents [9,15]. Additionally, Serogroup B has been shown to be the predominant serotype in carriage studies conducted in Europe and the United States (U.S.) [18][19][20]. Conversely, the most recent meningococcal disease outbreak was reported in a South Korean army basic training center in 2011, and this case was of serogroup W. Thereafter, an MCV4 vaccination program was implemented for all new military recruits in South Korea [21].
Despite the previous studies on seroprevalence and carriage rates of N. meningitidis in the South Korean population, the necessity of vaccinating specific populations such as college students is not well-defined. A study conducted in the U.S. showed that the overall incidence of meningococcal disease among university students was comparable to that of the general population; however, the disease rates among students living in dormitories were higher compared to those living off campus. Consequently, the Advisory Committee on Immunization Practices and the Committee on Infectious Diseases of the American Academy of Pediatrics recommended meningococcal conjugate vaccination for incoming college freshmen [22,23]. Although meningococcal vaccines are not included in the national immunization program for children in South Korea, MCV4 is currently available and recommended by the KCDC for immunocompromised individuals, travelers to endemic areas, laboratory workers, new military recruits, and university freshmen who plan to live in a dormitory [10,21]. Since South Korea has a conscription system, male adults must enlist in the military and receive MCV4. About 300,000 new recruits are estimated to be vaccinated annually; however, since male freshmen tend to enter military service after attending school for more than a year, herd immunity is not formed in the dormitory. While asymptomatic carriage rates do not necessarily indicate the potential for invasive disease, our study suggests that it is necessary to consider meningococcal vaccination for South Korean students before dormitory admission, given the importance of maintaining herd immunity.
The cultured isolates in this study were susceptible to most antibiotics, including ceftriaxone, ciprofloxacin, and rifampin, which are used as prophylactic antibiotics for contacts. However, 63.4% of the isolates showed low susceptibility to penicillin, which is recommended as a first-line drug in IMD. These results were similar to those of a previous study conducted in South Korea, in which 78% of the isolates showed low penicillin-susceptibility [9]. In the aforementioned study, penicillin resistance was reported in serogroups W and C. Although serogroup W was not isolated in our study, resistance was reported in all serogroups, including B; therefore, the spread of resistance should be a concern.
Our study has some limitations. First, the results may not represent the epidemiology of meningococcal carriage among university students in South Korea, since this study was performed on a small number of subjects within a university campus. There may also be some selection bias, as only students who signed informed consent were enrolled in the study. Moreover, seasonal variation could not be evaluated, since the study was performed between March and June. In addition, recall bias may affect the results, as we collected data based on the subjects' responses to the questionnaire. Although the same genotype propagation was determined based on MLST, this study was limited as whole-genome sequencing was not implemented and other epidemiologic grounds were not provided.
In conclusion, N. meningitidis carriage rates among new university entrants who lived in a dormitory in South Korea significantly increased during the first semester, probably due to the transmission of an identical genotype among students. Based on the risk of meningococcal disease, meningococcal vaccination should be considered for students before dormitory admission.
Supporting information S1 File. This is the questionnaire for study participants in Korean. (