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Carriage of Neisseria meningitidis and Neisseria lactamica among ethnic Greek school children from Russian immigrant families in Athens
~;D~~f~OOGY
AND
MICROBIOLOGY
ELSEVIER
FEMS Immunology and Medical Microbiology 23 (1999) 13-20
Carriage of Neisseria meningitidis and Neisseria lactamica among ethnic Greek school children from Russian immigrant families in Athens Jenny Kremastinou a,*, Georgina Tzanakaki a, Emmanuel Velonakis a, Aliki Voyiatzi a, Alexandra Nickolaou b, Robert A. Elton ‘, Donald Weir d, Caroline Blackwell d a National Meningitis Reference Laboratory, Athens School of Public Health, 196 Alesandras Ave., Athens, Greece ’ Department of Pediatrics. Pentelis Children’s Hospital, Athens, Greece ’ Medical Statistics Unit, University of Edinburgh, Edinburgh, UK ’ Department of Medical Microbiology, Universitv of Edinburgh, Edinburgh, UK
Received 22 July 1998; received in revised form 7 October 1998; accepted 7 October 1998
Abstract During February and March 1995, a survey of meningococcal carriage in 625 school children was carried out in a suburb of Athens in which there was a large number of ethnic Greeks who had immigrated from Russia beginning in the early 1990s. The objectives of the study were: (1) to determine if factors associated with carriage of meningococci observed in a previous study of Greek school children were similar for the immigrant population; (2) to compare phenotypic characteristics of meningococci from the immigrant population with those isolated from children in Athens. Overall isolation rate for meningococci was 82/625 (13.1%), significantly higher than that found for school children in Athens (5.8%) during the winter of 1990-1991 (5.8%) (x2 = 25.98, P = 0.0000003‘~ By univariate analysis, carriage was not associated with sex, number of individuals per household, blood group, secretor status, socioeconomic level or maternal smoking; however, it was associated with fathers’ smoking. The high proportion of men who smoked compared with the low proportion of women smokers might contribute to this finding. The main serogroup of meningococci isolated from this population was A (28%). While serogroup A appears to be more prevalent among Russian and Kurdish immigrants (14%) than among Greek school children or military recruits (4%), there has not been an increase in group A meningococcal disease in Greece. The isolation rate for N. lactamica was high 105/625 (17.3%). A few of these strains bound some of the monoclonal antibodies used for meningococcal serotyping and subtyping, and they are being examined in greater detail. 0 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords:
Immigrant
population:
Neisseria nteningitidis; Neisseria lactamica; Parental
smoking;
Serogroup
A
* Corresponding author. Tel.: +30 (1) 646 5982; Fax: +30 (1) 643 2258. 0928-8244/ 99/ $19.00 0 1999 Federation PII: SO928-8244(98)001 12-6
of European
Microbiological
Societies. Published
by Elsevier Science B.V. All rights reserved.
14
J. Krenzastinou
et al. I FEMS
bnmrmology
1. Introduction The influx of immigrants from countries of the former Soviet Union has raised questions regarding introduction of meningococci with antigenic types not usually found in western European populations. The return of a significant number of ethnic Greeks from the Soviet Union provided an opportunity to examine an immigrant group which was genetically the same as the indigenous Greek population. Following World War II and the Greek Civil War, large numbers of ethnic Greeks moved to the Soviet Union where they resided until the recent political changes which resulted in less stringent immigration and travel restrictions. Since the early 1990s immigrants (mainly from the Kazakhstan region) have been returning to the Athens area where they now live in relatively isolated communities in a suburb of the city. In this study there were two major objectives: (1) to determine if the factors associated with carriage of meningococci observed in a previous study of Greek school children [1] were associated with carriage of these bacteria in an immigrant population that did not differ significantly in genetic background from that of Athens; (2) to assess the phenotypic characteristics of strains of meningococci carried among the immigrant population to determine if these differed from those found in previous studies of Greek school children [2,3].
2. Subjects and methods 2. I. Populations examined During February and March 1995 a total of 625 pupils in three schools in an area approximately 15 km from Athens were screened for carriage of Neisseria meningitidis and Neisseria lactarnica. Family incomes are low, and in many families both mother and father work as market traders. Pupils in three primary schools in the area in which the majority of the immigrants live were surveyed. There were 169 pupils in school A, 242 in school B and 214 in school C. In addition to a throat swab, blood and saliva specimens were obtained. Permission for the studies
and Medical
Microbiology
23 (1999)
13-N
was obtained from the Ministry of Health and Ministry of Education: written informed consent for each child to participate in the survey was obtained from a parent or guardian. The parent or guardian filled in a questionnaire similar to that used in our previous studies of carriage among Greek school children. The questionnaire was used to obtain information on: place of birth and educational level of both parents; smoking habits of members of the household; number of residents in the household; age and sex of the child; place of birth; number of years resident in Greece; recent symptoms of respiratory tract infection. As in the previous studies, socioeconomic status was assessed by the numbers of years of education and level of education obtained by the parents: 0 = no formal schooling; 1 = demotico or primary school (6 years); 2 = lykio or middle school (7-9 years); 3 = gymnasio or secondary school (9-12 years); 4 = technical training; 5 = university [l]. The information was coded to insure confidentiality and entered into a database. ABO and Lewis blood groups were determined by agglutination with monoclonal reagents used in previous studies (Scottish National Blood Transfusion Service) and secretor status by ELISA for H, Lewisa and Lewis” antigens [1,46]. 2.2. Isolation and churucterisation of Neisseria species Pharyngeal swabs were cultured on modified New York City Medium and Neisseria species identified by Gram stain, oxidase reaction and biochemical characteristics (Gallerie Pasteur). Serogroups of the isolates were assessed by agglutination with polyclonal antisera (Wellcome Diagnostics) and serotypes and subtypes were determined by whole cell ELISA with monoclonal antibodies (RIVM, Bilthoven, The Netherlands) and described previously [3]. The same method was used to test a new serotype ‘22’ monoclonal antibody provided by the National Institute of Public Health, Prague, Czech Republic [7]. 2.3. Statistical analyses The x2 test was used to compare results for different groups with the Epi-info package.
1s
J. Kremastinou et al. I FEMS Immunology and Medicul Microbiology 23 (1999) 13-20
3. Results 3.1. Characteristics of’ the population The three primary schools had pupils aged 6-15, the older being recent arrivals who were learning Greek before attending secondary school (Fig. 1). For the analysis, immigrant families were defined as those in which the mother of the child was born in Russia. Of the 62.5 children in the study, 402 (64%) were from imrnigrant families. In school A 147/169 (87.5%) of pupils had mothers born in Russia compared with 1561242 (64%) in school B and 991 214 (46%) in school c’. Family sizes ranged from two to 16 persons per household and were larger for the immigrant families compared with local families, particularly among those with five or more persons in the household (x” = 40.69, df = 5, P = 0.00000) (Fig. 2). Among the parents of the children in the three schools, the educational level for those born in Russia was significantly higher: mothers, x2 = 84.34, df=4, P = 0.00000; fathers, x” = 85.72, df = 4, P=O.OOOOO(Figs. 3 an.d 4). The proportion of smokers among the mothers born in Russia was significantly lower 44/402 (11%) than among mothers born in Greece 55/219 (25%) (x” = 6.79, df= 1, P< 0.009). Smoking among fathers born in Russia (2631397, 66%) was slightly higher than that among fathers born in Greece (132/224, 59%) but the difference
6
7
8
9
4
5
7
6
=-I8
No. per household Fig. 2. Comparison Russian immigrants
of numbers of individuals and local families.
per household
for
was not significant. Smoking among fathers born in Russia was not significantly associated with educational level; however, among fathers born in Greece, the proportion of smokers decreased with increasing years of education (x2 = 14.2, df= 5, PzO.014). For Russian-born mothers, there was a higher proportion of smokers among those with fewer years of education (x’ = 9.09, df=4, P = 0.058). There was, however, no association between smoking and educational level among mothers born in Greece in either this study or our previous survey of families in Athens (Fig. 5).
10
Educational
Age Fig. I. Age distribution
3
of FLussian immigrant
and local children.
Fig. 3. Comparison and local mothers.
of educational
level
levels of Russian
immigrant
J. Kremastinou et al. I FEMS
16
Itnmunolog~v and Medical Microbiology
23 (1999) 13-20
pared with local (34/229, 14.8%) children. The proportion of local children from whom N. lactamica was isolated was higher (49/229, 21.4%) compared with that from immigrant children (56/396, 14.1%). There was no difference in isolation of meningococci from local or immigrant children in any of the three schools, but isolation of N. lactamica was significantly higher among local (26/89, 22.6%) compared with immigrant (7/92, 7%) children in school C (x’ = 8.69, P < 0.005). 3.3. Factors associated with isolation of Neisseria species
Educational Fig. 4. Comparison and local fathers.
of educational
level levels of Russian
There was no association between isolation of meningococci or N. lactamica and ABO blood group, secretor status, sex of the child or symptoms of respiratory tract infection.
immigrant
3.2. Isolation of Neisseria species 3.3.1.
Overall
isolation rates for meningococci and N. lactamica were 82/625 (13.1%) and 1051625 (17.3%) respectively. Information was incomplete for two of the children from whom N. meningitidis was isolated and from three from whom N. lactamica was isolated. These were not included in the following analyses in relation to carriage but all isolates were assessed for phenotypic characteristics. There was no significant difference in isolation of meningococci from immigrant (46/396, 11.6%) com-
H Athens
/0
% smokers
Russia
Family size
There was no clear trend between number of members of the household and isolation of either species. The majority of isolates were obtained from children from households with four, five or six members (Fig. 6). 3.3.2.
Age
Isolation of both meningococci and N. lactamica in relation to age reflected the age distribution of the
I
40 35 30 25 20 15 10
1
2
3
4
3
5
of smoking habits among Russian immigrant
5
6
7
>I8
No. per household
Educational category Fig. 5. Comparison and local mothers.
4
Fig. 6. Comparison of isolation of N. nteningitidis and N. lactato the number of individuals per household.
mica in relation
J. Kremstinou
et al. IFEMS Immunology and Medical Microbiology
children in the study. Although N. luctamica appeared to be found more often in the younger children compared with meningococci, the differences were not significant (Fig. 7).
11
70 60
3.3.3. Parental smoking Isolation of meningococci was not associated with mothers’ smoking but. was significantly associated with fathers’ smoking (x2 = 5.38, P= 0.020, OR = 2.66, 95% CI 1.13, 6.48). There was no association between parental smoking and isolation of A? luctamica from children. Smoking by members of the family other than the mother or father was noted for 7180 (8.75%) carriers of meningococci but only 4/104 (3.8%) carriers of N. hzctamica. 3.4. Characteristics of meningococcal N. lactamica isolates
23 (I9991 13-20
50
.
Or A
B
I C
NO
serogroup Fig. 8. Comparison of serogroups of meningococcal isolates from Russian immigrant and local school children with meningococcal isolates from Athenian children.
and
3.4. I. N. meningitidis The majority of the isolates (39182, 47.5%) were not serogroupable. The most common serogroup was A (23/82, 28%), followed by B (12182, 14.2”/0) and C (4182, 4.8%) (Fig. 8). The group A isolates were isolated predom.inantly from children whose mother and/or father were born in Russia (17/23, 74%) and 6/23 (26%) from children whose parents were born in mainland Greece or the Greek islands.
Nearly half the group A isolates (11123, 48%) were obtained from children in school C; seven serogroup A isolates were from the 99 immigrant children (7%) and four from the 214 local children (2%) (x2 = 3.98, P < 0.05). The proportion of non-serotypable isolates was 36/ 82 (44%) and the most common serotypes were 15 (15182, 18.3%) 22 (11182, 13.4%) 4 (11/82, 13.4%)
45 40 35 30 %
25 20 15 10 5 0 1
6
7
8
9
10
11
12
13
14
2a
2b
4
14
15
22
nt
15
serotype Age Fig. 7. Comparison of isolation of N. meningitidis and N. mica in relation to the age of the school children.
lacta-
Fig. 9. Comparison of serotypes of meningococcal isolates from Russian immigrant and local school children with meningococcal isolates from Athenian children.
J. Kremastinou et al. IFEMS
18
Immwolog~v and Medical Microbiolog_v 23 (1999)
and 14 (g/82, 11%) (Fig. 9). The most common subtypes were 5 (16/82, 19.5%), 7 (14/82, 17%) and 1 (9/ 82, 11%). 3.4.2. N. lactamica None of the N. lactamica isolates was agglutinated by serogroup antisera. There were a few isolates that reacted with some serotype and subtype antibodies. They were retested for their biochemical characteristics to confirm they were N. lactamica and are being further characterised in other studies.
4. Discussion 4.1. Isolation of Neisseria species from immigrant and local school children
The proportion of carriers in this study (13.1%) was significantly higher compared with that found in our previous survey of 1038 primary and secondary school children in Athens during the winter of 1990-1991 (5.8%) (x2 = 25.98, P=O.O000003). In addition to a higher proportion of meningococci isolated in this study, there was a high carriage rate of N. lactamica (16.8%). As in our previous study of Greek school children, by univariate analyses, carriage of meningococci was not associated with sex, ABO blood group, secretor status, symptoms of respiratory tract infection or number of members of the household. In contrast to the Stonehouse survey [8] and a longitudinal study in Belgium [9], carriage rates were not associated with lower socioeconomic status. This was observed in our previous study of meningococcal carriage in school children in two areas of Athens that differed significantly in two markers for socioeconomic status, housing costs and educational level of the parents [l]. Although the immigrant parents were significantly better educated than the parents born in Greece, there was no difference in the proportion of N. meningitidis or N. lactamica isolated from children whose mothers were born in Greece compared with those whose mothers were born in Russia. In our previous study, we found that regardless of educational level. about one third of the mothers in the two areas of Athens were smokers [l]. The pro-
13-20
portion of women smokers was lower in both the local and immigrant groups, 25% and 10% respectively. Maternal smoking and smoking by members of the household other than the father were significant factors associated with carriage of meningococci in the study of Athens school children [l]; however, in this study, carriage was not associated with maternal smoking but with smoking by the father. Parental smoking was not associated with isolation of N. lactamica. Exposure of children to cigarette smoke is an important risk factor for meningococcal disease and carriage [lO,l I]. Among Greek recruits, active smoking was significantly associated with carriage of meningococci [5]. Epithelial cells from smokers bound significantly more N. meningitidis and N. lactamica than cells from non-smokers [12]. Studies on isolation of meningococci and N. lactamica from Scottish infants and their mothers found that infants whose mothers are carriers of these bacteria were more likely to be colonised by these species and they were more likely to be smokers [13]. Shepherd [14] suggested that a non-smoker who lives with a smoker will be more likely to develop a respiratory tract infection. Smokers are more likely to have respiratory infections, and we have demonstrated that several respiratory viruses (respiratory syncytial virus and influenza A) enhance binding of meningococci and N. lactamica to an epithelial cell line [15,16]. N. lactamica was isolated from less than 1% of Greek military recruits (unpublished observations). If fathers who are smokers are more heavily colonised by meningococci, this could explain the association between fathers’ smoking and isolation of meningococci but not N. lactamica. 4.2.
Characteristics
of the Neisseria isolates
Serogroup A is responsible for epidemics of meningitis in several parts of the world including Russia [ 171, but it has not been a common cause of meningococcal disease in Greece over the past 10 years [3,18]. Compared with the proportion of serogroup A among meningococcal isolates obtained in our previous studies of carriage in both school children (4%) and young adults (4%) the proportion of serogroup A among isolates from the population in the
J. Kremastinou et al. I FEMS
Itnmunology and Medical Microbiology
present study was 28%. Serogroup A was also found in 5/35 (14%) of meningococcal isolates from a group of 163 Kurdish immigrants (21% carriage rate) screened in a recent study [19]. Despite the presence of increased carriage rates of meningococci and a higher proportion of serogroup A in both these immigrant populations, serogroup A accounted for only 21194 (1%) isolates from cases of meningococcal disease sent to the reference laboratory in Athens between 1993 and 1997. The finding that some of the N. lactamica isolates reacted with serotype or subtype reagents is being further investigated. This has not been observed for over 60 N. luctamica isolates from British populations examined by the Scottish reference laboratory (Dr. L.E. Smart, personal communication) or among isolates of N. luctamica from Albanian children provided by Dr. E. Mastori, Institute of Hygiene and Epidemiology. Tirana, Albania. In conclusion, our findings indicate that there is a significantly higher carriage rate of N. meningitidis in the population examined which contained a high proportion of ethnic Greeks who are recent immigrants from Russia. There was also a higher than expected carriage rate of N. luctamicu in both immigrant and local children. In contrast to our previous findings for school children in Athens, maternal smoking was not a significant risk factor for carriage of meningococci but fathers’ smoking was. As carriage of meningococci is significantly higher among smokers, the high proportion of smokers among the fathers and the lower levels of smoking among women in both the immigrant and local population might contribute to this difference. In relation to our findings of a low level of (carriage of N. luctumicu among adult males (l&35), we suggest that the source of these bacteria in the children is probably from mothers and younger siblings. The predominant serogroup among isolates obtained from this population was A. Although this serogroup was found in both immigrant and local children and also in Kurdish immigrants, it is not associated with an increase in disease due to serogroup A in Greece. These findings indicate that introduction of meningococcal strains by immigrant groups does not necessarily lead to increased incidence of disease in the indigenous population.
23 (19991 13-20
19
Acknowledgments This work was supported by the Greek Ministry Health and in part by the Meningitis Association Scotland.
of of
References 111 Kremastinou, J., Tzanakaki, G., Blackwell, CC., Kallergi, C., Chronas, G., Mentis. A. and Weir, D.M. (1994) Parental smoking and carriage of Neisseria meningitidis among Greek school children, Stand. J. Infect. Dis. 26. 719-723. PI Tzanakaki, G., Blackwell, C.C., Kremastinou, J., Kallergi, C. and Weir, D.M. (1992) Antibiotic sensitivities of meningococcal isolates obtained from patients and carriers in Greece. Epidemiol. Infect. 108, 449-456. [31 Tzanakaki. G., Blackwell, C.C., Kremastinou. J., Kallergi, C., Fallon, R.J and Weir, D.M. (1993) Serogroups, serotypes and subtypes of Neisseria meningitidis isolated from patients and carriers in Greece. .I. Med. Microbial. 38. 19-22. 141 Rahat, A., Stewart, J., Blackwell. C.C. and Weir, D.M. (1990) A semi-quantitative method for measurement of H type 1 and H type 2 on buccal cells and in saliva of secretors and nonsecretors. Vox Sang. 59. 101-105. [51 Blackwell, C.C., Tzanakaki, G., Kremastinou, J., Weir, D.M., Vakalis. N.. Elton. R.A and Mentis, A. (1992) Factors affecting carriage of Nrisseria nmingitidis among Greek military recruits. Epidemiol. Infect. 108, 441448. [61 Blackwell. C.C., Weir, D.M.. James, V.S. et al. (1990) Secretor status, smoking and carriage of Neisseria nzeningitidis. Epidemiol. Infect. 104, 203-209. 171 Tzanakaki, G.. Kriz, P., Kremastinou, J., Musilek, M., Smart, L.E. and Blackwell C.C. (1997) Reactivity of the new monoclonal antibody ‘22’ with meningococcal strains isolated from patients and carriers in Greece. FEMS Immunol. Med. Microbiol. 19. 1-5. K.A.V., Stuart, J.M.. Jones, D.M. and Noah, [81 Cartwright. N.D. (1987) The Stonehouse survey. Nasopharyngeal carriage of meningococci and Neisseria lactamica. Epidemiol. Infect. 99, 591-601. 191 de Wals, P., Gilquin, C., DeMaeyer, S., Bouckaert, A., Noel, A., Lechat. M.F. and Lafontaine, A. (1983) Longitudinal study of asymptomatic carriage in two Belgian populations of school children, J. Infect. 6, 1477156. UOI Stuart. J.M., Cartwright, K.A.V., Dawson, J.A., Rickard, J. and Noah, N.D. (1988) Risk factors for meningococcal disease: a case control study in south west England. Commun. Med. 2, 139-145. [ill Haneberg. B., Tonjum, T.. Rodahl, K. and Gedde-Dahl, T.W. (1983) Factors preceding the onset of meningococcal disease with special emphasis on passive smoking. stressful events, physical fitness and general symptoms of ill health. NIPH Ann. 6, 169-174.
20
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et al. I FEMS Itnnumology and Medical Microbiology 23 (1999) 13-20
[12] El Ahmer, O.R.. Essery. SD.. Saadi, A.T., Ogilvie. M.M., Weir, D.M. and Blackwell, C.C. (1998) The effect of cigarette smoking on adherence of respiratory pathogens to buccal epithelial cells FEMS Immunol. Med. Microbial. (in press). 23. 21-36. [13] Mackenzie, D.A.C., James, V.S., Elton. R.A. et al. (1996) Toxigenic bacteria and SIDS: nasopharyngeal flora in the first year of life. Fourth SIDS International, pp. 166-167. [14] Shepherd. R.J. (1992) Respiratory irritation from environmental tobacco smoke. Arch. Environ. Health 47, 30&305. [15] Raza, M.W.. Ogilvie, M.M.. Blackwell, CC., Stewart, J.. Elton, R.A. and Weir, D.M. (1993) Effect of respiratory syncytial virus infection on binding of Neisseria meningitidis and Haemophihrs inflrrenzar type b to a human epithelial cell line (HEp-2). Epidemiol. Infect. 110, 339-347. [16] El Ahmer, O.R., Raza, M.W.. Ogilvie, M.M., Blackwell. C.C., Weir, D.M. and Elton. R.A. (1996) The effect of respiratory
virus infection on expression of cell surface antigens associated with binding of potentially pathogenic bacteria. In: Toward Anti-Adhesin Therapy of Microbial Diseases (Ofek, I. and Kahane. I.. Eds.). pp. 1699177. Plenum, New York. [17] Achtman. M., Kusecek. B.. Morelli. G.A. et al. (1992) Comparison of the variable antigens expressed by clone IV-l and subgroup III of Neisseria meningitidis serogroup A. J. Infect. Dis. 165. 53-68. [18] Kremastinou. J.. Tzanakaki. G., Pagalis, A. et al. (1998) Recent emergence of serogroup C meningococcal disease in Greece FEMS Immunol. Med. Microbial. (in press). 23, 4955. [19] Kremastinou. .I., Tzanakaki. G., Weir, D.M. and Blackwell, C.C. (1997) Carriage rates for different populations in Greece and characteristics of meningococcal isolates. 4th European Monitoring Group on Meningococci, Paris. France p. 45.
AND
MICROBIOLOGY
ELSEVIER
FEMS Immunology and Medical Microbiology 23 (1999) 13-20
Carriage of Neisseria meningitidis and Neisseria lactamica among ethnic Greek school children from Russian immigrant families in Athens Jenny Kremastinou a,*, Georgina Tzanakaki a, Emmanuel Velonakis a, Aliki Voyiatzi a, Alexandra Nickolaou b, Robert A. Elton ‘, Donald Weir d, Caroline Blackwell d a National Meningitis Reference Laboratory, Athens School of Public Health, 196 Alesandras Ave., Athens, Greece ’ Department of Pediatrics. Pentelis Children’s Hospital, Athens, Greece ’ Medical Statistics Unit, University of Edinburgh, Edinburgh, UK ’ Department of Medical Microbiology, Universitv of Edinburgh, Edinburgh, UK
Received 22 July 1998; received in revised form 7 October 1998; accepted 7 October 1998
Abstract During February and March 1995, a survey of meningococcal carriage in 625 school children was carried out in a suburb of Athens in which there was a large number of ethnic Greeks who had immigrated from Russia beginning in the early 1990s. The objectives of the study were: (1) to determine if factors associated with carriage of meningococci observed in a previous study of Greek school children were similar for the immigrant population; (2) to compare phenotypic characteristics of meningococci from the immigrant population with those isolated from children in Athens. Overall isolation rate for meningococci was 82/625 (13.1%), significantly higher than that found for school children in Athens (5.8%) during the winter of 1990-1991 (5.8%) (x2 = 25.98, P = 0.0000003‘~ By univariate analysis, carriage was not associated with sex, number of individuals per household, blood group, secretor status, socioeconomic level or maternal smoking; however, it was associated with fathers’ smoking. The high proportion of men who smoked compared with the low proportion of women smokers might contribute to this finding. The main serogroup of meningococci isolated from this population was A (28%). While serogroup A appears to be more prevalent among Russian and Kurdish immigrants (14%) than among Greek school children or military recruits (4%), there has not been an increase in group A meningococcal disease in Greece. The isolation rate for N. lactamica was high 105/625 (17.3%). A few of these strains bound some of the monoclonal antibodies used for meningococcal serotyping and subtyping, and they are being examined in greater detail. 0 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords:
Immigrant
population:
Neisseria nteningitidis; Neisseria lactamica; Parental
smoking;
Serogroup
A
* Corresponding author. Tel.: +30 (1) 646 5982; Fax: +30 (1) 643 2258. 0928-8244/ 99/ $19.00 0 1999 Federation PII: SO928-8244(98)001 12-6
of European
Microbiological
Societies. Published
by Elsevier Science B.V. All rights reserved.
14
J. Krenzastinou
et al. I FEMS
bnmrmology
1. Introduction The influx of immigrants from countries of the former Soviet Union has raised questions regarding introduction of meningococci with antigenic types not usually found in western European populations. The return of a significant number of ethnic Greeks from the Soviet Union provided an opportunity to examine an immigrant group which was genetically the same as the indigenous Greek population. Following World War II and the Greek Civil War, large numbers of ethnic Greeks moved to the Soviet Union where they resided until the recent political changes which resulted in less stringent immigration and travel restrictions. Since the early 1990s immigrants (mainly from the Kazakhstan region) have been returning to the Athens area where they now live in relatively isolated communities in a suburb of the city. In this study there were two major objectives: (1) to determine if the factors associated with carriage of meningococci observed in a previous study of Greek school children [1] were associated with carriage of these bacteria in an immigrant population that did not differ significantly in genetic background from that of Athens; (2) to assess the phenotypic characteristics of strains of meningococci carried among the immigrant population to determine if these differed from those found in previous studies of Greek school children [2,3].
2. Subjects and methods 2. I. Populations examined During February and March 1995 a total of 625 pupils in three schools in an area approximately 15 km from Athens were screened for carriage of Neisseria meningitidis and Neisseria lactarnica. Family incomes are low, and in many families both mother and father work as market traders. Pupils in three primary schools in the area in which the majority of the immigrants live were surveyed. There were 169 pupils in school A, 242 in school B and 214 in school C. In addition to a throat swab, blood and saliva specimens were obtained. Permission for the studies
and Medical
Microbiology
23 (1999)
13-N
was obtained from the Ministry of Health and Ministry of Education: written informed consent for each child to participate in the survey was obtained from a parent or guardian. The parent or guardian filled in a questionnaire similar to that used in our previous studies of carriage among Greek school children. The questionnaire was used to obtain information on: place of birth and educational level of both parents; smoking habits of members of the household; number of residents in the household; age and sex of the child; place of birth; number of years resident in Greece; recent symptoms of respiratory tract infection. As in the previous studies, socioeconomic status was assessed by the numbers of years of education and level of education obtained by the parents: 0 = no formal schooling; 1 = demotico or primary school (6 years); 2 = lykio or middle school (7-9 years); 3 = gymnasio or secondary school (9-12 years); 4 = technical training; 5 = university [l]. The information was coded to insure confidentiality and entered into a database. ABO and Lewis blood groups were determined by agglutination with monoclonal reagents used in previous studies (Scottish National Blood Transfusion Service) and secretor status by ELISA for H, Lewisa and Lewis” antigens [1,46]. 2.2. Isolation and churucterisation of Neisseria species Pharyngeal swabs were cultured on modified New York City Medium and Neisseria species identified by Gram stain, oxidase reaction and biochemical characteristics (Gallerie Pasteur). Serogroups of the isolates were assessed by agglutination with polyclonal antisera (Wellcome Diagnostics) and serotypes and subtypes were determined by whole cell ELISA with monoclonal antibodies (RIVM, Bilthoven, The Netherlands) and described previously [3]. The same method was used to test a new serotype ‘22’ monoclonal antibody provided by the National Institute of Public Health, Prague, Czech Republic [7]. 2.3. Statistical analyses The x2 test was used to compare results for different groups with the Epi-info package.
1s
J. Kremastinou et al. I FEMS Immunology and Medicul Microbiology 23 (1999) 13-20
3. Results 3.1. Characteristics of’ the population The three primary schools had pupils aged 6-15, the older being recent arrivals who were learning Greek before attending secondary school (Fig. 1). For the analysis, immigrant families were defined as those in which the mother of the child was born in Russia. Of the 62.5 children in the study, 402 (64%) were from imrnigrant families. In school A 147/169 (87.5%) of pupils had mothers born in Russia compared with 1561242 (64%) in school B and 991 214 (46%) in school c’. Family sizes ranged from two to 16 persons per household and were larger for the immigrant families compared with local families, particularly among those with five or more persons in the household (x” = 40.69, df = 5, P = 0.00000) (Fig. 2). Among the parents of the children in the three schools, the educational level for those born in Russia was significantly higher: mothers, x2 = 84.34, df=4, P = 0.00000; fathers, x” = 85.72, df = 4, P=O.OOOOO(Figs. 3 an.d 4). The proportion of smokers among the mothers born in Russia was significantly lower 44/402 (11%) than among mothers born in Greece 55/219 (25%) (x” = 6.79, df= 1, P< 0.009). Smoking among fathers born in Russia (2631397, 66%) was slightly higher than that among fathers born in Greece (132/224, 59%) but the difference
6
7
8
9
4
5
7
6
=-I8
No. per household Fig. 2. Comparison Russian immigrants
of numbers of individuals and local families.
per household
for
was not significant. Smoking among fathers born in Russia was not significantly associated with educational level; however, among fathers born in Greece, the proportion of smokers decreased with increasing years of education (x2 = 14.2, df= 5, PzO.014). For Russian-born mothers, there was a higher proportion of smokers among those with fewer years of education (x’ = 9.09, df=4, P = 0.058). There was, however, no association between smoking and educational level among mothers born in Greece in either this study or our previous survey of families in Athens (Fig. 5).
10
Educational
Age Fig. I. Age distribution
3
of FLussian immigrant
and local children.
Fig. 3. Comparison and local mothers.
of educational
level
levels of Russian
immigrant
J. Kremastinou et al. I FEMS
16
Itnmunolog~v and Medical Microbiology
23 (1999) 13-20
pared with local (34/229, 14.8%) children. The proportion of local children from whom N. lactamica was isolated was higher (49/229, 21.4%) compared with that from immigrant children (56/396, 14.1%). There was no difference in isolation of meningococci from local or immigrant children in any of the three schools, but isolation of N. lactamica was significantly higher among local (26/89, 22.6%) compared with immigrant (7/92, 7%) children in school C (x’ = 8.69, P < 0.005). 3.3. Factors associated with isolation of Neisseria species
Educational Fig. 4. Comparison and local fathers.
of educational
level levels of Russian
There was no association between isolation of meningococci or N. lactamica and ABO blood group, secretor status, sex of the child or symptoms of respiratory tract infection.
immigrant
3.2. Isolation of Neisseria species 3.3.1.
Overall
isolation rates for meningococci and N. lactamica were 82/625 (13.1%) and 1051625 (17.3%) respectively. Information was incomplete for two of the children from whom N. meningitidis was isolated and from three from whom N. lactamica was isolated. These were not included in the following analyses in relation to carriage but all isolates were assessed for phenotypic characteristics. There was no significant difference in isolation of meningococci from immigrant (46/396, 11.6%) com-
H Athens
/0
% smokers
Russia
Family size
There was no clear trend between number of members of the household and isolation of either species. The majority of isolates were obtained from children from households with four, five or six members (Fig. 6). 3.3.2.
Age
Isolation of both meningococci and N. lactamica in relation to age reflected the age distribution of the
I
40 35 30 25 20 15 10
1
2
3
4
3
5
of smoking habits among Russian immigrant
5
6
7
>I8
No. per household
Educational category Fig. 5. Comparison and local mothers.
4
Fig. 6. Comparison of isolation of N. nteningitidis and N. lactato the number of individuals per household.
mica in relation
J. Kremstinou
et al. IFEMS Immunology and Medical Microbiology
children in the study. Although N. luctamica appeared to be found more often in the younger children compared with meningococci, the differences were not significant (Fig. 7).
11
70 60
3.3.3. Parental smoking Isolation of meningococci was not associated with mothers’ smoking but. was significantly associated with fathers’ smoking (x2 = 5.38, P= 0.020, OR = 2.66, 95% CI 1.13, 6.48). There was no association between parental smoking and isolation of A? luctamica from children. Smoking by members of the family other than the mother or father was noted for 7180 (8.75%) carriers of meningococci but only 4/104 (3.8%) carriers of N. hzctamica. 3.4. Characteristics of meningococcal N. lactamica isolates
23 (I9991 13-20
50
.
Or A
B
I C
NO
serogroup Fig. 8. Comparison of serogroups of meningococcal isolates from Russian immigrant and local school children with meningococcal isolates from Athenian children.
and
3.4. I. N. meningitidis The majority of the isolates (39182, 47.5%) were not serogroupable. The most common serogroup was A (23/82, 28%), followed by B (12182, 14.2”/0) and C (4182, 4.8%) (Fig. 8). The group A isolates were isolated predom.inantly from children whose mother and/or father were born in Russia (17/23, 74%) and 6/23 (26%) from children whose parents were born in mainland Greece or the Greek islands.
Nearly half the group A isolates (11123, 48%) were obtained from children in school C; seven serogroup A isolates were from the 99 immigrant children (7%) and four from the 214 local children (2%) (x2 = 3.98, P < 0.05). The proportion of non-serotypable isolates was 36/ 82 (44%) and the most common serotypes were 15 (15182, 18.3%) 22 (11182, 13.4%) 4 (11/82, 13.4%)
45 40 35 30 %
25 20 15 10 5 0 1
6
7
8
9
10
11
12
13
14
2a
2b
4
14
15
22
nt
15
serotype Age Fig. 7. Comparison of isolation of N. meningitidis and N. mica in relation to the age of the school children.
lacta-
Fig. 9. Comparison of serotypes of meningococcal isolates from Russian immigrant and local school children with meningococcal isolates from Athenian children.
J. Kremastinou et al. IFEMS
18
Immwolog~v and Medical Microbiolog_v 23 (1999)
and 14 (g/82, 11%) (Fig. 9). The most common subtypes were 5 (16/82, 19.5%), 7 (14/82, 17%) and 1 (9/ 82, 11%). 3.4.2. N. lactamica None of the N. lactamica isolates was agglutinated by serogroup antisera. There were a few isolates that reacted with some serotype and subtype antibodies. They were retested for their biochemical characteristics to confirm they were N. lactamica and are being further characterised in other studies.
4. Discussion 4.1. Isolation of Neisseria species from immigrant and local school children
The proportion of carriers in this study (13.1%) was significantly higher compared with that found in our previous survey of 1038 primary and secondary school children in Athens during the winter of 1990-1991 (5.8%) (x2 = 25.98, P=O.O000003). In addition to a higher proportion of meningococci isolated in this study, there was a high carriage rate of N. lactamica (16.8%). As in our previous study of Greek school children, by univariate analyses, carriage of meningococci was not associated with sex, ABO blood group, secretor status, symptoms of respiratory tract infection or number of members of the household. In contrast to the Stonehouse survey [8] and a longitudinal study in Belgium [9], carriage rates were not associated with lower socioeconomic status. This was observed in our previous study of meningococcal carriage in school children in two areas of Athens that differed significantly in two markers for socioeconomic status, housing costs and educational level of the parents [l]. Although the immigrant parents were significantly better educated than the parents born in Greece, there was no difference in the proportion of N. meningitidis or N. lactamica isolated from children whose mothers were born in Greece compared with those whose mothers were born in Russia. In our previous study, we found that regardless of educational level. about one third of the mothers in the two areas of Athens were smokers [l]. The pro-
13-20
portion of women smokers was lower in both the local and immigrant groups, 25% and 10% respectively. Maternal smoking and smoking by members of the household other than the father were significant factors associated with carriage of meningococci in the study of Athens school children [l]; however, in this study, carriage was not associated with maternal smoking but with smoking by the father. Parental smoking was not associated with isolation of N. lactamica. Exposure of children to cigarette smoke is an important risk factor for meningococcal disease and carriage [lO,l I]. Among Greek recruits, active smoking was significantly associated with carriage of meningococci [5]. Epithelial cells from smokers bound significantly more N. meningitidis and N. lactamica than cells from non-smokers [12]. Studies on isolation of meningococci and N. lactamica from Scottish infants and their mothers found that infants whose mothers are carriers of these bacteria were more likely to be colonised by these species and they were more likely to be smokers [13]. Shepherd [14] suggested that a non-smoker who lives with a smoker will be more likely to develop a respiratory tract infection. Smokers are more likely to have respiratory infections, and we have demonstrated that several respiratory viruses (respiratory syncytial virus and influenza A) enhance binding of meningococci and N. lactamica to an epithelial cell line [15,16]. N. lactamica was isolated from less than 1% of Greek military recruits (unpublished observations). If fathers who are smokers are more heavily colonised by meningococci, this could explain the association between fathers’ smoking and isolation of meningococci but not N. lactamica. 4.2.
Characteristics
of the Neisseria isolates
Serogroup A is responsible for epidemics of meningitis in several parts of the world including Russia [ 171, but it has not been a common cause of meningococcal disease in Greece over the past 10 years [3,18]. Compared with the proportion of serogroup A among meningococcal isolates obtained in our previous studies of carriage in both school children (4%) and young adults (4%) the proportion of serogroup A among isolates from the population in the
J. Kremastinou et al. I FEMS
Itnmunology and Medical Microbiology
present study was 28%. Serogroup A was also found in 5/35 (14%) of meningococcal isolates from a group of 163 Kurdish immigrants (21% carriage rate) screened in a recent study [19]. Despite the presence of increased carriage rates of meningococci and a higher proportion of serogroup A in both these immigrant populations, serogroup A accounted for only 21194 (1%) isolates from cases of meningococcal disease sent to the reference laboratory in Athens between 1993 and 1997. The finding that some of the N. lactamica isolates reacted with serotype or subtype reagents is being further investigated. This has not been observed for over 60 N. luctamica isolates from British populations examined by the Scottish reference laboratory (Dr. L.E. Smart, personal communication) or among isolates of N. luctamica from Albanian children provided by Dr. E. Mastori, Institute of Hygiene and Epidemiology. Tirana, Albania. In conclusion, our findings indicate that there is a significantly higher carriage rate of N. meningitidis in the population examined which contained a high proportion of ethnic Greeks who are recent immigrants from Russia. There was also a higher than expected carriage rate of N. luctamicu in both immigrant and local children. In contrast to our previous findings for school children in Athens, maternal smoking was not a significant risk factor for carriage of meningococci but fathers’ smoking was. As carriage of meningococci is significantly higher among smokers, the high proportion of smokers among the fathers and the lower levels of smoking among women in both the immigrant and local population might contribute to this difference. In relation to our findings of a low level of (carriage of N. luctumicu among adult males (l&35), we suggest that the source of these bacteria in the children is probably from mothers and younger siblings. The predominant serogroup among isolates obtained from this population was A. Although this serogroup was found in both immigrant and local children and also in Kurdish immigrants, it is not associated with an increase in disease due to serogroup A in Greece. These findings indicate that introduction of meningococcal strains by immigrant groups does not necessarily lead to increased incidence of disease in the indigenous population.
23 (19991 13-20
19
Acknowledgments This work was supported by the Greek Ministry Health and in part by the Meningitis Association Scotland.
of of
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[12] El Ahmer, O.R.. Essery. SD.. Saadi, A.T., Ogilvie. M.M., Weir, D.M. and Blackwell, C.C. (1998) The effect of cigarette smoking on adherence of respiratory pathogens to buccal epithelial cells FEMS Immunol. Med. Microbial. (in press). 23. 21-36. [13] Mackenzie, D.A.C., James, V.S., Elton. R.A. et al. (1996) Toxigenic bacteria and SIDS: nasopharyngeal flora in the first year of life. Fourth SIDS International, pp. 166-167. [14] Shepherd. R.J. (1992) Respiratory irritation from environmental tobacco smoke. Arch. Environ. Health 47, 30&305. [15] Raza, M.W.. Ogilvie, M.M.. Blackwell, CC., Stewart, J.. Elton, R.A. and Weir, D.M. (1993) Effect of respiratory syncytial virus infection on binding of Neisseria meningitidis and Haemophihrs inflrrenzar type b to a human epithelial cell line (HEp-2). Epidemiol. Infect. 110, 339-347. [16] El Ahmer, O.R., Raza, M.W.. Ogilvie, M.M., Blackwell. C.C., Weir, D.M. and Elton. R.A. (1996) The effect of respiratory
virus infection on expression of cell surface antigens associated with binding of potentially pathogenic bacteria. In: Toward Anti-Adhesin Therapy of Microbial Diseases (Ofek, I. and Kahane. I.. Eds.). pp. 1699177. Plenum, New York. [17] Achtman. M., Kusecek. B.. Morelli. G.A. et al. (1992) Comparison of the variable antigens expressed by clone IV-l and subgroup III of Neisseria meningitidis serogroup A. J. Infect. Dis. 165. 53-68. [18] Kremastinou. J.. Tzanakaki. G., Pagalis, A. et al. (1998) Recent emergence of serogroup C meningococcal disease in Greece FEMS Immunol. Med. Microbial. (in press). 23, 4955. [19] Kremastinou. .I., Tzanakaki. G., Weir, D.M. and Blackwell, C.C. (1997) Carriage rates for different populations in Greece and characteristics of meningococcal isolates. 4th European Monitoring Group on Meningococci, Paris. France p. 45.