Carriage of Neisseria species in communities with different ra tes of meningococcal d isease

A single clone, Neisseria meningitidis serogroup C (C:2a:P1.2), was isolated from seven patients during a cluster of cases of meningococcal disease in Ontario in 1989. To determine whether the clone was present in asymptomatic individuals in the same population, pharyngeal swabs were taken from 7% (644 of 9125) of residents who were vaccinated during the outbreak. Rates of isolation of Neisseria species were also compared to those in two other geographical areas which did not have an elevated incidence of meningococcal disease. The rate of carriage of N meningitidis in the asymptomatic individuals sampled was between 1.9% and 5.4%. The clone isolated from patients was not present among the carrier strains as determined by sero- and subtyping and electrophoretic analysis of metabolic enzymes. Age greater than six years was the only factor associated with colonization with N meningitidis.

T HE PRESENCE OF NEISSERIA MENlNGITIDIS IN THE UPPER respiratory tract is not usually associated with disease.The occasional acquisition of virulent strains of N meningitidis in this anatomical area is thought to be critical for the development of disease in s usceptible individuals (1,2).
During the winter of 1988-89.an increase in the number of cases of meningococcal disease occurred in the province of Ontario (3).Within that period.nine clinical or culture-confirmed cases of meningococcal disease occurred in a predominantly rural county (Victoria County) comprising three townships and two towns surrounding Lindsay.Ontario.Census figures from 1986 estimated the total population of the area to be 28,135.The overall rate of meningococcal disease in the 'zero to 19 years' age group was 75 per 100.000;however.the rate was 198 per 100,000 in the "10 to 14 years' age group, and 50 per 100.000 in the "five to nine years• age group.Of seven culture-confirmed cases.all had N meningitidis serogroup C. serotype 2a.subtype Pl.2 (C:2a:Pl.2) (4).
A study examining nasopharyngeal carriage of Neisseria species was undertaken to determine whether the outbreak strain of N meningitidis serogroup C was prevalent in the disease-free population of Victoria County.Two other control areas were sampled to determine whether carriage of the same clone was present in regions that did not have an elevated incidence of meningococcal disease.ln the control areas .the authors also attempted to identify risk factors associated with carriage of Neisseria s pec ies.

SUBJECTS AND METHODS
The neisseria carriage study was done in Victoria County (the area with an elevated incidence of disease) and Perth County, Ontario and in three health units in sou them Alberta (areas that did not have increased numbers of patients with meningococcal disease).
The culture survey was done during an immunization program with quadrivalent vaccine (A.C.Y.W-135) for persons aged six months to 18 years in U1e Victoria County town of Lindsay.Ontario in February 1989.Cultures were obtained just prior to receipt of meningococcal vaccine.A total of644 of9125 people (7%) who presented for vaccination h ad throat cultures done .Information on age, sex and treatment with rifampin in the two weeks prior to swabbing was obtained from subjects or their parents.
Perth County, surrounding Stratford.Ontario, is 270 Ian southwest of Victoria County.In this area, a convenience sample of schools was used, and children were selected using a table of random numbers from class lists provided by the schools.Questionnaires were sent home for completion.To select children below school age.the following two methods of recruitment were used.A random sample was tak en of U1e 30% who attended licensed daycare facilities .For the 70% not Carriage of Neisseria species attending these facilities.recruitm ent involved children born after 1984 who presented to major or group medical practices in the a rea during the time swabbing took place.If U1e accompanying parent agreed .s he or h e completed U1e questionnaire and the child was swabbed.
The control area of the Lethbridge.Barons-Eureka-Warner a nd Chinook Health Units located in southern Alberta h as sin1ilar demographics to the two Ontario areas and is located 3500 km west of Victoria County.Students in all schools in the tl1ree health units were randomly selected.Questionnaires and consent form s were mailed to the students' homes and returned to the school.Preschool children were selected randomly from routine in1munization clinics in the healtl1 units where all preschool in1n1Linizations are done in Alberta.
Questionnaires and consent forms were completed at the immunization clinics.Swabbing in the control areas was completed within an eight week period.Exclusion criteria for participation was any cu rrent acute illness or fever.
Subjects in the two control areas were asked about age.sex.symptoms of respiratory illness and antibiotic use in the preceding two weeks.and history of serious or sign ificant medical problems.Information was also obtained on family size, attendance at school, daycare.play groups.team sports and clubs.as well as parent education level.
The health units notified parents who had requested results of U1e culture.Parents were free to discuss results wit!1 their private physicians.Treatment of carriers was not recommended.Laboratory methods: Swabbing consisted of rubbing U1e posterior pharynx behind the uvula with a cotton-Upped swab .In the two Ontario areas.swabs were inoculated directly onto New York City medium in JEM -BEC plates.Carbon dioxide-generating tab lets were added to the plates.which were then put in p lastic bags.In Alberta.U1e swabs were inoculated onto plates witl1 chocolate agar and modified Thayer-Martin medium and placed in Bio-Bag carbon dioxide transport systems (Marion Scientific.Missouri).Plates were incubated at 37°C witl1in 2 h of swabbing using portable or local hospital incubators and examined after 24 to 48 h of incubation.Identification of Neisseria species.seragrouping.serotyping and subtyping were carried out as described previously (5-8).Monoclonal antibodies for sero-and subtyping were kindly supplied by Dr JT   Atlanta.Georgia).Mantcl-Haenszel statistics were u sed to establish associations between the dis ease ancl ris k factors.Relative risks and 95% confidence in tervals were a lso calculated us ing the STATCALC program in EPIINFO .r is her•s exact test was used when expected cell frequencies in a 2x2 ta ble were less than five.Two-tailed probabilities were used to establis h s ignificance.
62 RESULTS Carriage rate: The carriage ra tes for all Neisser•ia s pecies in the three areas s urveyed ra nged between 13.8% and 17. 1% (Table 1).SouU1ern Alberta had a s ta Us Ucally lower carriage ra te fo r N meningilidis U1an boU1 areas in Ontarto.
In the th ree areas.age was the only factor consistently associated with colonization wi th N meningilidis.Age-adj usted carriage rates in person s younger than six years old were eight per 1000 a nd 241 per I 000 for N meningilidis and Neisseria lactamica.respectively.The rate of carriage for persons six years or older was 53 per 1 000 for both N meningilidis and N laclam ica.The majority of isolates of N men ingilidis were obtained from individuals between 12 and 18 years of age in a ll th ree areas: VictOJi a County 30 of 35 or 86% : PcrU1 County 24 of 34 or 7 1%: and Alber ta 12 of 15 o r 80%.
The following facto rs were not associated wi th an inc reased risk of colon iZation wiU1 eitl1er N meningilidis or N lactamica: symptom s of respira tory t.r acl infections in the previous two weeks: underlying medical illness: number of persons living in the househ old: ed ucational level of parents; and daycare altendance o r participa• tion in organized clubs or s po rts.In Victoria County.rifampin had been used by 23 of 493 respondents to the questionnai re.A significant.associaUon (two-tailed) was not fo und between rifampin use and carriage of any Neisseria species.but statis tical power using U1cse numbers was less than I 0% and was.therefo re.insuf• ficient to detect a s ignificant association if il exists.

Serogroups, s erotypes and subtypes of N menin• g i tidis:
The dis tribution of N meningil idis by scrogroup is s hown in Table 2 .Serogroup C and Y st ra ins were isola ted mor e freq uenUy in Victo ria County.
The dis tribution of sero-and subtypes among sero• grou ps of N meningitidis is s hown in Table 3. Overall.17 combinations of serotypes and/or subtypes were found am ong U1e carrier strains.T he mos t common serotypes, 4 and 15. were associated with 26.4% and 15.7%.respectively. of all strains.Nea rly one-halfoflhe s trains were nonserot:ypeable .Serotype 4 was asso• elated mainly With serogrou p 8 and nong rou pable s trains.while serotype 15 was fo und most frequently a mong nongroupable strains.
The frequency of s u btypes was as follows: Pl.l (9.6%).Pl.2 (22 .9%).PJ.3 (3.6%).P l.7 (6.0%) and ' P l .l 6 (7.2o/o) .Just under 50% of the s tra in s failed to react with the sub typing monoclonal antiboclies .Fifteen of the 19 strains containing the Pl.2 epitope were isolated in Victoria County.Fourteen of these strains were isolated from carriers aged 14 t.o 18 years; the 15th strain was isolated from a 26-year-old carrier.The Pl.2 epitope occurred on three C:2a, one C:2b, one Y:2c.six Y:NT and fo u r NG:NT strains isolated in Victoria County.Only two strains (Y:NT:Pl.2) from Perth County and two strains (NG :NT:Pl.2and 29e:NT:Pl.2) from southern Alberta contained the Pl.2 epitope.Elec tr oph o re tic analysis o f enzymes: Enzyme electrophoretic analysis of the seven serogroup C carrier strains revealed that.none was the same enzyme electrophoretic type as that causing the focal outbreak of disease in the Victoria County area.Enzyme electrophoretic patterns of representative strains ofY:NT:Pl.2and NG:NT:Pl.2isolated in the Victoria County area were identical to each other but were clissimilar from all serogroup C strains including that causing the outbreak.

DI SCUSSION
The overall carriage rates for N meningitidis in this study were consistent with carriage rates of between l% and 10% in other similar surveys (1,11.12).The difference in carriage rate between southern Alberta and Ontario was probably not due to differences in technique since comparable age-specific isolation rates for N Lactamica were obtained.Carriage rates of the same serogroup of N meningitidis in household contacts and military recruits can be between 30% and 45% (11.13).The present study a lso reconfirmed that carriage of N meningitidis is more common in children over the age of six years .especially teenagers.while carriage of N Lactamica is associated with younger age groups (14).Other factors associated with increased risk of carriage and/or clisease have been crowded sleeping areas, upper respiratory tract symptoms.and coincident respiratory viral and mycoplasmal agents (13.15.16).
The unique aspects of th is stu dy are that it compared community carriage and clonality of group C meningococci in geographically separated outbreak and nonoutbreak areas .In England.outbreak strains of group B meningococci were found more commonly among carriers in housing estates with a higher incidence of disease.but other areas within the san1e city also had colonization with the same subtypes of organism (17).A Canadian carriage study was carried out in an isolated Arctic community with a high incidence of group B clisease but comparisons between the disease-causing isolates and the carrier strains were not reported (18).Another random carrier study took place in the Port Hope and Cobourg areas of Ontario in September 1989.In that area the carriage rate for N meningitidis was 4% (54 of 1326).One inclividual had a group C isolate wh ich was non typeable and unrelated to the outbreak strain accorcling to enzyme electrophoretic analysis (personal comm unication).
The lack of carriage of the clone causing the outbreak in the Victoria County area is not unexpected.Very low carriage rates of outbreak strains of group B disease have been reported in England and Norway (19.20) .Factors that cou ld contribute to decreased carriage of outbreak strains in asymptomatic persons may include a shorter period of colon ization or a rapid onset of clinical disease fo llowing acquis ition of the organism in nonirnmune hosts.Indeed.Edwards et al (21) have shown U1at carriage of group C strains causing invasive disease in military recruits is less than two weeks prior to development of clinical symptoms .Others have suggested that strains causing outbreaks or epidemics may be more virulent than strains respons ib le for sporadic disease (1,2).Proof of U1is wou ld require longitudinal stuclies comparing morbiclity and mortality associated with different strains of meningococci.
It. appears that asymptomatic carriage may be associated with a diversity of serogroups and sera/subtypes of meningococcal strains in contrast to only a few sero/subtypes such as 2a:Pl.2.2b:Pl.2.2b:Pl.Ham.4:Pl.l5 and l5:Pl.l6,which have caused the majority of outbreaks of group Band C disease (7,10.19,[22][23][24].In this study.none of the 12 combinations of serotype/nonserot.ypeand/or subtypes identified among the 49 strains isolated in the two geographical areas free from disease was of U1e sera/subtypes mentioned above.Although not all carrier strains were examined using enzyme electrophoretic analysis.diversity in genetic relatedness has previously been shown to be greater in N meningitidis strains colonizing carriers compared to isolates from patients with meningococcal clisease (25).
Acquired immunity to meningococcal clisease depends in part on prior colonization with microorganisms in order to develop antibodies to group-specific polysaccharide and/or other cell surface components such as outer membrane proteins and lipopolysaccharides (lipo-oligosaccharides). Thus.an individual may be at greater risk of disease if not previously colonized with N meningitidis or N lactamica or oU1er cross-reacting organisms (14 ,26).For N lactamica.the existence of common epitopes to meningococcal lipopolysaccharides may provide cross-immunity against meningococcal d isease (27).In this study.15 of 19 meningococcal strains (78.9%) containinO" the Pl.2 epitope were isolated from carriers in U1e Victoria County.Wh ile it is unknown if U1ese particular carriers had antiboclies to the Pl.2 epitope it is conceivable U1at they had developed immunity against group C organisms through carriage ofnongroup C meningococci that were immunologically related through outer membrane proteins.Introduction of a strain of N meningitidisca..rrying epitopes into a subgroup of a population not previously exposed to a proportion of lhose antigens may partially explain the occu rrence of localized outbreaks.
Techniques such as sero-and s u btyping and enzyme electrophoretic typing have permitted lhe a uthors to establ ish lhat a homogeneo us clone ca used th e outbreak in the Victoria Co un ty a rea a n d U1 at lhe gro up C A CKNOWLEDGEMENTS: We thank Dr J Spika and Ms E Paul on for reviewing this manuscript.We also thank the taff and tuclenls in the communities in the participating health units and their affiliated laboratorie for their assistance.We thank Dr JT Pootman and Dr WD Zollinger for supplying monoclonal antibodies.and Fran\oisc Collins and Linda Mancino at the LCDC for their excellent laboratory expertise.ca rri er strains were infrequent.h eterogen ous and of differing s u btypes and clon es.By identifYing clones of paU1ogen ic meningococci th at have cau sed ou tbreaks.it may be possible to look for virulen ce factors that ch a racterize these organis ms and to h elp cla rify U1 e co m plexities of men ingococcal d isease outbreaks .

REFERENCES
Poolman of the National Institute of Public Health and Environmental Protection in the Netherlands.and Dr WD Zollinger of U1e Walter Reed Army Institute in Washington.DC.Electrophoretic analysis of metabolic enzymes was carried out as described by Selander et al (9) and Caugant et al (10).Distributio n o f Neisseria species p resent in sele c ted asymptomatic populations Data analysis: Data analysis was carried out using EPIINFO version 3 (Division of Disease Surveillance and Epidemiologic Studies.Centers for Disease Control, TABLE 1'No Neisserto species 1soloted 1 1ncluded f1ve stroms of Neisseria perftova;slcca and f1ve stra1ns of Neisseria polysocchorea