JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1992, p. 386-390 0095-1137/92/020386-05$02.00/0 Copyright C 1992, American Society for Microbiology
Vol. 30, No. 2
Molecular Epidemiology of Haemophilus influenzae Type b in The Gambia HENK A. BIJLMER,l.2t* LOEK VAN ALPHEN,2 LEA GEELEN-VAN DEN BROEK,2 BRIAN M. GREENWOOD,1 HANS A. VALKENBURG,3 AND JACOB DANKERT2 Medical Research Council Laboratories, Fajara, The Gambia,1 Department of Medical Microbiology and WHO Reference Laboratory for Bacterial Meningitis RIVM, University of Amsterdam,2 and Department of Epidemiology, Erasmus University of Rotterdam,3 The Netherlands Received 25 July 1991/Accepted 29 October 1991
One hundred two invasive and 64 noninvasive isolates of Haemophilus influenzae were collected in the course of a 2-year prospective field study on the epidemiology of H. influenzae meningitis in The Gambia. The isolates were serotyped, biotyped, and subtyped by outer membrane protein (OMP) profile analysis (OMP subtyping). H. influenzae meningitis was found to be caused by serotype b (95%). In invasive disease, serotype a, although present in the throat of healthy children, caused only occasionally (5.9%) disease. The distribution of biotypes of H. influenzae appeared to be very similar to that found outside The Gambia. A distinct pattern of OMP subtypes, different from other parts of the world, is prevalent in H. influenzae type b (Hib) in The Gambia. OMP subtypes 2, 4, 5, 8, and 9 were observed to be predominant. These subtypes, except subtype 2, have not been described. L subtypes (subtypes 2, 4, and 8) were associated with invasive disease, whereas non-L subtypes (subtypes 5 and 9) were found more often in healthy carriers (P < 0.001). A significant difference in geographical distribution was found in subtypes of noninvasive Hib strains (P < 0.05). We conclude that in The Gambia H. influenzae invasive disease is caused mainly by type b strains with a limited number of OMP subtypes, which are different from the subtypes found elsewhere in the world. These data are important for the surveillance of Hib disease in developing countries and are baseline data for a Hib polyribosyl-ribitolphosphate-conjugated vaccine trial in The Gambia. Alternative Hib OMP vaccines should include a set of representative OMPs.
Haemophilus influenzae meningitis is a serious infectious disease in childhood with a high morbidity and mortality rate. The majority of invasive disease is caused by H. influenzae serotype b (Hib) strains. The incidence of H. influenzae meningitis varies widely between populations in different parts of the world. In populations with a high incidence of H. influenzae meningitis, the peak incidence of infection occurs at a younger age than that in populations with a low incidence. In a developing country such as The Gambia, the incidence of H. influenzae meningitis, 297 cases per 100,000 children under 1 year of age per year, is high, and the age at which the highest incidence occurs, 5 months old, is low (5). Several Hib-conjugated vaccine trials in industrialized areas showed satisfactory results (6, 9). In nonindustrialized areas two Hib-conjugated vaccine studies have been carried out; one, the Navajo study, was successful (20), and the other, the Alaskan study, in which a different Hib-conjugated vaccine was used, showed an unsatisfactory efficacy rate (32). Accurate epidemiological data from developing countries for use as background for vaccine efficacy studies are scarce. Serotyping, biotyping, outer membrane protein (OMP) subtyping and lipopolysaccharide (LPS) serotyping are valuable tools in characterizing H. influenzae (23). OMP subtyping has shown to be one of the most discriminatory methods for epidemiological purposes. Two distinct OMP subtyping
systems have been developed. Barenkamp et al. (2) have set OMP subtyping system which is used mainly in the United States. Van Alphen et al. have described a slightly different sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) system (29) which has been used in
up an
several European OMP subtyping studies (25, 28). Twentydifferent OMP subtypes have been described in Hib isolates from the United States (3), of which 5 account for 85% of all isolates from Hib invasive disease. In Western Europe one subtype, subtype 1 (3L), is predominant (25). The distribution of OMP subtypes of Hib isolates in the Far East shows a different pattern from the subtypes identified in the United States (18, 34). Epidemiological data on subtypes of Hib from Africa are lacking. A prospective field study and several carriage surveys were carried out to get a better understanding of the epidemiology of H. influenzae meningitis in the Gambia (4, 5). In this paper we describe the molecular epidemiology of H. influenzae isolates on the basis of capsular polysaccharide serotyping, biotyping, and OMP subtyping. The implications of these data for vaccination are discussed. one
MATERIALS AND METHODS
Geography. The Gambia is a small West African country stretching along the river Gambia over a length of 350 km. The country is nowhere more than 40 km wide. The whole country is encompassed by Senegal, except where it borders the Atlantic Ocean. There is one main road on the south
Corresponding author. t Present address: Department of Medical Microbiology, Bronovo Hospital, Bronovolaan 5, 2597 AX Den Haag, The Netherlands. *
bank and one on the north bank of the river. The Gambia lies in the sub-Sahel zone. There is one rainy season, from July 386
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VOL. 30, 1992
to the end of October, and usually there is no rainfall outside this period. Colection, culture, and storage of strains. During the course of a 2-year prospective field study on the epidemiology of H. influenzae meningitis in the western and middle part of The Gambia, H. influenzae was isolated from cerebrospinal fluid (CSF) from meningitis patients (5). In addition, H. influenzae was isolated from other specimens on clinical indication. Each patient was represented by a single isolate. Hib carrier strains were collected from healthy carriers during several surveys done in different parts of the country, including Basse in the East of the country, 350 km upriver; Brefet, 60 km upriver (middle); Brikama and Lamin, 20 km from the coast (west) (4). Primary isolation of invasive strains of H. influenzae was on chocolate agar and on horse blood agar. H. influenzae was identified by testing dependency for X-factor in the delta-amino levulinic acid test as described by Kilian (13) and V-factor dependency by means of a satellite test on a NAD-deficient plate. Hib strains of carriers were isolated from healthy children on selective culture plates containing sheep anti-Hib antiserum as described previously (4). Immediately after initial isolation all strains were stored at -70°C on glass beads as suspensions in broth and supplemented with 15% glycerol, till further processing. Characterization of isolates. (i) Serotyping. Serotyping of isolates was done by using a latex agglutination test, as described by Dirks-Go (8), with polyclonal rabbit antisera specific for type a to f. After absorption, no cross-reactivity between the serotypes a to f could be detected. A set of reference H. influenzae strains serotype a to f were used as controls. (ii) Biotyping. The ability of the isolates to produce indole, urease, and/or ornithine decarboxylase was tested as described by Kilian (14). Strains of biotype I, III, and V were used as controls in each test. (iii) OMP subtyping. Isolation of cell envelopes and analysis of the OMP composition by SDS-PAGE was done essentially as described by Lugtenberg et al. (17). The process of cell disruption was automated as described by Van Alphen et al. (30). The OMP patterns of the isolates were compared with those of the OMP subtype reference strains of Hib used in the Van Alphen system (29) and the 23 OMP subtype reference strains used in the Barenkamp system (3). L (light) subtypes and non-L subtype strains, as described in the nomenclature of Barenkamp (2), were differentiated in a whole-cell enzyme-linked immunosorbent assay (ELISA), as described by Abdillahi and Poolman (1), by using murine monoclonal antibody (MAb) 5HA5. MAb 5HA5 reacts only with the L subtypes of the Barenkamp collection of strains (26). (iv) LPS serotyping. LPS serotype 1 was determined by means of a whole-cell ELISA as described by Abdillahi and Poolman (1) by using MAb 5HD9 (26, 27). Statistical analysis. A two-tailed Fisher exact test was used for the analysis.
RESULTS Characterization of isolates. A total of 166 strains of H.
influenzae have been characterized. One hundred
two
strains of all serotypes were from patients with invasive disease (Table 1) and 64 Hib strains originated from the throats of healthy people and were obtained during carrier surveys. Most of the known Hib clinical syndromes were
387
TABLE 1. Distribution of capsular serotypes from H. influenzae causing invasive disease in relation to the sites of origin No. of isolates in serotype:
Source (no. of isolates)
a
b
c to f
NEa
Blood (32) CSF (60) Pleural fluid (5) Synovial fluid (1) Pus (4)
3 2 0 0 1
26 57 4 1 2
0 0 0 0 0
3 1 1 0
6
90
0
6
Total (102) a
1
NE, nonencapsulated.
seen in The Gambia, with the notable exception of epiglottitis. The serotype distribution of the isolates from invasive disease according to the site of origin is shown in Table 1. Meningitis was caused by serotype b in 95% of the cases. Of all invasive strains 88% belonged to type b; 6 strains (6%) were serotype a. Six isolates were nonencapsulated. The 154 Hib isolates were analyzed further. Biotyping of the Hib isolates revealed that biotype I was the predominant type among the invasive strains (84%) as well as among noninvasive strains (77%). Biotype II accounted for 12 of 90 (13%) Hib invasive strains and 15 of 64 (23%) noninvasive strains. Biotypes other than I or II were rarely encountered among invasive isolates. The distribution of OMP subtypes of the 90 Hib isolates from invasive disease is shown in Table 2. Subtype 4 was predominant (54%). The 64 throat isolates belonged to various OMP subtypes (Table 2), with the exception of subtype 2. Subtype 2 is identical to subtype 2L in the Barenkamp system and has been described previously among European and U.S. isolates (2, 28). Subtypes 4, 5, 8, and 9 are unique. They were not identical to any of the subtypes described by Van Alphen or Barenkamp (2, 3, 10, 29). These subtypes were assigned as new numbers in the Van Alphen subtyping system. The profile of the Gambian subtypes is shown in Fig. 1. All isolates of OMP subtypes 2, 4, and 8 reacted with MAb 5HA5, indicating that they were L type. None of the strains with subtype 5 and 9 strains reacted with MAb 5HA5. Since LPS serotype 1 has been associated with invasive disease (27), all invasive disease and noninvasive disease TABLE 2. Distribution of OMP subtypes of Hib from patients with invasive disease and from carriers in the general population in The Gambia Source (no. of isolates)
No. of isolate in subtype: 2
4
5
8
9
NSAa
Blood (26) CSF (57) Pleural fluid (4) Synovial fluid (1) Pus (2) Total invasive (90)
4 3 0 0 0 7
10 34 3 0 2 49
1 5 0 1 0 7
2 5 0 0 0 7
2 2 0 0 0 4
7 8 1 0 0 16
Throat, noninvasive (64)
0
19
20
3
10
12
7
68
27
10
14
28
Total (154) a
NSA, no subtype assigned.
*_.
BIJLMER ET AL.
388
J. CLIN. MICROBIOL.
370
1000 24589
MW24589
9468-*Wp
a
"
........
_ o
W
20 subtypes (numbers at top) in nonheated specimens; 8 through 12, the same specimens modified by heat. On the vertical axis, right side, are shown the positions of the OMPs a to e; on the left side are shown molecular mass markers (in kilodaltons) and the position of the heat-modifiable OMPs a' and d' in unheated form.
Hib isolates were LPS serotyped for serotype 1. Only 1 of 90 invasive disease isolates were positive, as were 7 of 64 of the carrier strains from the general population. Epidemiology. OMP subtype 2 was isolated exclusively from patients with invasive disease (Table 2). OMP subtype S was isolated less frequently from invasive disease isolates than from healthy carriers in the general population (Fisher exact test, P < 0.001). A similar result was obtained for OMP subtype 9 (Fisher exact test, P < 0.05). OMP subtype 4 was found more frequently in invasive disease than in healthy throats (Fisher exact test, P < 0.01). The distribution of OMP subtype 8 and the nontypeable strains was similar for patients and healthy carriers. A significant correlation was found between L subtypes (subtype 2, 4, and 8) and invasiveness of Hib strains; non-L types (subtypes 5 and 9) were found more frequently among carriers (Fisher exact test, P < 0.001). The geographical distribution of OMP subtypes isolated from carriers in the general population shows clear differences (Table 3). OMP subtype 2 was not isolated from carriers in any of the three locations. Subtype 9 was not found in carriers living in Basse. In contrast, subtype S was present almost exclusively in this location. The distribution of the various subtypes was random over the respective
TABLE 3. Geographical distribution of OMP subtypes of Hib obtained from throat swabs in the general population of The Gambia (n = 64) OMP subtype
2 4 5 8 9 Nonsubtypeable Total
No. of isolates from following village:
Brikama/Lamin (west)
Brefet (center)
Basse (east)
0 4 2 1 3 5
0
1
0 12 18 2 0 6
11
38
15
3 0 0 7
locations (Fisher exact test, P = nonsignificant), except for subtypes 5 and 9 (Fisher exact test, P < 0.05).
DISCUSSION Distribution of H. influenzae sero- and subtypes in The Gambia. Serotype b is the predominant H. influenzae serotype causing invasive diseases in most parts of the world. In the United States (10), The Netherlands (7), Denmark and Norway (15), Sweden (22), and Finland (19), around 99% of H. influenzae invasive disease is caused by this serotype. In the population with a very high incidence of H. influenzae invasive disease, the Alaskan Eskimos, serotype b is also the most frequently isolated serotype (33). We found that sero-
type b accounted for 95% of H. influenzae isolates from meningitis patients in The Gambia, which is slightly lower than the 99% reported by us on a smaller series (5). Our observations correlate well with the incidence of 96.7% serotype b for H. influenzae from meningitis patients in neighboring Senegal. A surprisingly high proportion of serotype a isolates from patients with invasive disease has been reported from nonindustrialized areas (12, 16, 31). Serotype a hardly occurred among CSF isolates from The Gambia but accounted for 5.8% of all invasive strains (Table 1). Serotype a strains were also present in the throats of healthy children (unpublished data). A higher proportion of serotype a in The Gambia was reported by Wall et al., but the number of strains analyzed was small (31). We conclude that in West Africa, as elsewhere, serotype b is responsible for most of the invasive diseases caused by encapsulated H. influenzae. The distribution of biotypes did not differ significantly from that in other reports (15, 22, 28, 33). In invasive H. influenzae disease, biotype I (84%) and biotype 11 (13%)
predominated. We have found five distinct OMP subtypes among the Gambian Hib isolates. These subtypes were compared to the OMP subtypes as described by Barenkamp et al. and Van Alphen et al. (2, 3, 25, 28). Only subtype 2, identical to the Barenkamp subtype 2L, has been described previously. OMP subtypes 4, 5, 8, and 9 are new subtypes and have been designated as new numbers in the OMP subtyping system described by Van Alphen et al. (25, 28). We conclude that in The Gambia Hib has a pattern of OMP profiles different from those in other parts of the world. OMP subtypes and invasive disease. It has been reported that certain OMP subtypes cause different types of disease and that the distribution of subtypes among carrier strains differs from that of disease isolates (21, 24). L subtypes (subtypes 2, 4, and 8) were associated with invasive disease, whereas non-L subtypes (subtypes 5 and 9) were found more
MOLECULAR EPIDEMIOLOGY OF Hib IN THE GAMBIA
VOL. 30, 1992
often in healthy carriers (P < 0.001) (Tables 2 and 3). The predominance of subtype 5 among carrier strains may be explained by differences in the geographical distribution (Table 3). Subtype 9 strains probably have a low virulence, since they were significantly more frequent among carrier isolates than disease isolates in the catchment area. The significant difference in the geographical distribution of noninvasive Hib strains between the western part and the eastern part of the country is in contrast with data on subtype distribution in the United States (10). We conclude that the area of circulation of bacterial isolates apparently is limited, although the transmission rate of carrier strains in The Gambia was shown to be rapid (4). LPS serotyping has been developed relatively recently as an additional epidemiological tool for studying Hib disease. In Western Europe (Finland, Sweden, Norway, Denmark, The Netherlands, Germany, the United Kingdom, and France), with the notable exception of Iceland, LPS serotype 1 is the most prevalent type in invasive disease (25, 27). In the United States, the LPS serotype distribution among invasive isolates is much more diverse. In our study, LPS serotype 1 was found infrequently (4.2%) and seven of eight LPS 1 strains were noninvasive Hib. LPS serotype 1 is therefore probably not a virulence determinant for invasive disease. As we did not test for LPS serotypes other than LPS serotype 1, we cannot be certain that the Hib isolates of The Gambia do not possess a common LPS type. Implications of the epidemiological data for vaccination. This study has shown that invasive and noninvasive isolates of Hib in The Gambia differ in several important characteristics from isolates studied in other areas in the world. The data are important for the surveillance of Hib disease in developing countries and are baseline data for a Hib PRPconjugated vaccine trial in The Gambia. If, however, the currently available PRP-conjugated vaccines prove to be less efficacious than we expect them to be in countries with epidemiology similar to that in The Gambia, a set of representative OMPs from Hib, which this study has shown to be different from those found in the western world, should be included in an alternative Hib OMP vaccine (11, 18). ACKNOWLEDGMENTS Our thanks are due to Abdulie Sanneh for his indefatigable efforts in carrying out the field study and to Sam Appiah, Ignatius Baldeh, Ramu Njie, and Kutube Manneh for their contribution as laboratory technicians. This work was supported by grant W93-122 from the Foundation for the Advancement of Tropical Research, the Netherlands. REFERENCES 1.
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Molecular Epidemiology of Haemophilus influenzae Type b in The Gambia HENK A. BIJLMER,l.2t* LOEK VAN ALPHEN,2 LEA GEELEN-VAN DEN BROEK,2 BRIAN M. GREENWOOD,1 HANS A. VALKENBURG,3 AND JACOB DANKERT2 Medical Research Council Laboratories, Fajara, The Gambia,1 Department of Medical Microbiology and WHO Reference Laboratory for Bacterial Meningitis RIVM, University of Amsterdam,2 and Department of Epidemiology, Erasmus University of Rotterdam,3 The Netherlands Received 25 July 1991/Accepted 29 October 1991
One hundred two invasive and 64 noninvasive isolates of Haemophilus influenzae were collected in the course of a 2-year prospective field study on the epidemiology of H. influenzae meningitis in The Gambia. The isolates were serotyped, biotyped, and subtyped by outer membrane protein (OMP) profile analysis (OMP subtyping). H. influenzae meningitis was found to be caused by serotype b (95%). In invasive disease, serotype a, although present in the throat of healthy children, caused only occasionally (5.9%) disease. The distribution of biotypes of H. influenzae appeared to be very similar to that found outside The Gambia. A distinct pattern of OMP subtypes, different from other parts of the world, is prevalent in H. influenzae type b (Hib) in The Gambia. OMP subtypes 2, 4, 5, 8, and 9 were observed to be predominant. These subtypes, except subtype 2, have not been described. L subtypes (subtypes 2, 4, and 8) were associated with invasive disease, whereas non-L subtypes (subtypes 5 and 9) were found more often in healthy carriers (P < 0.001). A significant difference in geographical distribution was found in subtypes of noninvasive Hib strains (P < 0.05). We conclude that in The Gambia H. influenzae invasive disease is caused mainly by type b strains with a limited number of OMP subtypes, which are different from the subtypes found elsewhere in the world. These data are important for the surveillance of Hib disease in developing countries and are baseline data for a Hib polyribosyl-ribitolphosphate-conjugated vaccine trial in The Gambia. Alternative Hib OMP vaccines should include a set of representative OMPs.
Haemophilus influenzae meningitis is a serious infectious disease in childhood with a high morbidity and mortality rate. The majority of invasive disease is caused by H. influenzae serotype b (Hib) strains. The incidence of H. influenzae meningitis varies widely between populations in different parts of the world. In populations with a high incidence of H. influenzae meningitis, the peak incidence of infection occurs at a younger age than that in populations with a low incidence. In a developing country such as The Gambia, the incidence of H. influenzae meningitis, 297 cases per 100,000 children under 1 year of age per year, is high, and the age at which the highest incidence occurs, 5 months old, is low (5). Several Hib-conjugated vaccine trials in industrialized areas showed satisfactory results (6, 9). In nonindustrialized areas two Hib-conjugated vaccine studies have been carried out; one, the Navajo study, was successful (20), and the other, the Alaskan study, in which a different Hib-conjugated vaccine was used, showed an unsatisfactory efficacy rate (32). Accurate epidemiological data from developing countries for use as background for vaccine efficacy studies are scarce. Serotyping, biotyping, outer membrane protein (OMP) subtyping and lipopolysaccharide (LPS) serotyping are valuable tools in characterizing H. influenzae (23). OMP subtyping has shown to be one of the most discriminatory methods for epidemiological purposes. Two distinct OMP subtyping
systems have been developed. Barenkamp et al. (2) have set OMP subtyping system which is used mainly in the United States. Van Alphen et al. have described a slightly different sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) system (29) which has been used in
up an
several European OMP subtyping studies (25, 28). Twentydifferent OMP subtypes have been described in Hib isolates from the United States (3), of which 5 account for 85% of all isolates from Hib invasive disease. In Western Europe one subtype, subtype 1 (3L), is predominant (25). The distribution of OMP subtypes of Hib isolates in the Far East shows a different pattern from the subtypes identified in the United States (18, 34). Epidemiological data on subtypes of Hib from Africa are lacking. A prospective field study and several carriage surveys were carried out to get a better understanding of the epidemiology of H. influenzae meningitis in the Gambia (4, 5). In this paper we describe the molecular epidemiology of H. influenzae isolates on the basis of capsular polysaccharide serotyping, biotyping, and OMP subtyping. The implications of these data for vaccination are discussed. one
MATERIALS AND METHODS
Geography. The Gambia is a small West African country stretching along the river Gambia over a length of 350 km. The country is nowhere more than 40 km wide. The whole country is encompassed by Senegal, except where it borders the Atlantic Ocean. There is one main road on the south
Corresponding author. t Present address: Department of Medical Microbiology, Bronovo Hospital, Bronovolaan 5, 2597 AX Den Haag, The Netherlands. *
bank and one on the north bank of the river. The Gambia lies in the sub-Sahel zone. There is one rainy season, from July 386
MOLECULAR EPIDEMIOLOGY OF Hib IN THE GAMBIA
VOL. 30, 1992
to the end of October, and usually there is no rainfall outside this period. Colection, culture, and storage of strains. During the course of a 2-year prospective field study on the epidemiology of H. influenzae meningitis in the western and middle part of The Gambia, H. influenzae was isolated from cerebrospinal fluid (CSF) from meningitis patients (5). In addition, H. influenzae was isolated from other specimens on clinical indication. Each patient was represented by a single isolate. Hib carrier strains were collected from healthy carriers during several surveys done in different parts of the country, including Basse in the East of the country, 350 km upriver; Brefet, 60 km upriver (middle); Brikama and Lamin, 20 km from the coast (west) (4). Primary isolation of invasive strains of H. influenzae was on chocolate agar and on horse blood agar. H. influenzae was identified by testing dependency for X-factor in the delta-amino levulinic acid test as described by Kilian (13) and V-factor dependency by means of a satellite test on a NAD-deficient plate. Hib strains of carriers were isolated from healthy children on selective culture plates containing sheep anti-Hib antiserum as described previously (4). Immediately after initial isolation all strains were stored at -70°C on glass beads as suspensions in broth and supplemented with 15% glycerol, till further processing. Characterization of isolates. (i) Serotyping. Serotyping of isolates was done by using a latex agglutination test, as described by Dirks-Go (8), with polyclonal rabbit antisera specific for type a to f. After absorption, no cross-reactivity between the serotypes a to f could be detected. A set of reference H. influenzae strains serotype a to f were used as controls. (ii) Biotyping. The ability of the isolates to produce indole, urease, and/or ornithine decarboxylase was tested as described by Kilian (14). Strains of biotype I, III, and V were used as controls in each test. (iii) OMP subtyping. Isolation of cell envelopes and analysis of the OMP composition by SDS-PAGE was done essentially as described by Lugtenberg et al. (17). The process of cell disruption was automated as described by Van Alphen et al. (30). The OMP patterns of the isolates were compared with those of the OMP subtype reference strains of Hib used in the Van Alphen system (29) and the 23 OMP subtype reference strains used in the Barenkamp system (3). L (light) subtypes and non-L subtype strains, as described in the nomenclature of Barenkamp (2), were differentiated in a whole-cell enzyme-linked immunosorbent assay (ELISA), as described by Abdillahi and Poolman (1), by using murine monoclonal antibody (MAb) 5HA5. MAb 5HA5 reacts only with the L subtypes of the Barenkamp collection of strains (26). (iv) LPS serotyping. LPS serotype 1 was determined by means of a whole-cell ELISA as described by Abdillahi and Poolman (1) by using MAb 5HD9 (26, 27). Statistical analysis. A two-tailed Fisher exact test was used for the analysis.
RESULTS Characterization of isolates. A total of 166 strains of H.
influenzae have been characterized. One hundred
two
strains of all serotypes were from patients with invasive disease (Table 1) and 64 Hib strains originated from the throats of healthy people and were obtained during carrier surveys. Most of the known Hib clinical syndromes were
387
TABLE 1. Distribution of capsular serotypes from H. influenzae causing invasive disease in relation to the sites of origin No. of isolates in serotype:
Source (no. of isolates)
a
b
c to f
NEa
Blood (32) CSF (60) Pleural fluid (5) Synovial fluid (1) Pus (4)
3 2 0 0 1
26 57 4 1 2
0 0 0 0 0
3 1 1 0
6
90
0
6
Total (102) a
1
NE, nonencapsulated.
seen in The Gambia, with the notable exception of epiglottitis. The serotype distribution of the isolates from invasive disease according to the site of origin is shown in Table 1. Meningitis was caused by serotype b in 95% of the cases. Of all invasive strains 88% belonged to type b; 6 strains (6%) were serotype a. Six isolates were nonencapsulated. The 154 Hib isolates were analyzed further. Biotyping of the Hib isolates revealed that biotype I was the predominant type among the invasive strains (84%) as well as among noninvasive strains (77%). Biotype II accounted for 12 of 90 (13%) Hib invasive strains and 15 of 64 (23%) noninvasive strains. Biotypes other than I or II were rarely encountered among invasive isolates. The distribution of OMP subtypes of the 90 Hib isolates from invasive disease is shown in Table 2. Subtype 4 was predominant (54%). The 64 throat isolates belonged to various OMP subtypes (Table 2), with the exception of subtype 2. Subtype 2 is identical to subtype 2L in the Barenkamp system and has been described previously among European and U.S. isolates (2, 28). Subtypes 4, 5, 8, and 9 are unique. They were not identical to any of the subtypes described by Van Alphen or Barenkamp (2, 3, 10, 29). These subtypes were assigned as new numbers in the Van Alphen subtyping system. The profile of the Gambian subtypes is shown in Fig. 1. All isolates of OMP subtypes 2, 4, and 8 reacted with MAb 5HA5, indicating that they were L type. None of the strains with subtype 5 and 9 strains reacted with MAb 5HA5. Since LPS serotype 1 has been associated with invasive disease (27), all invasive disease and noninvasive disease TABLE 2. Distribution of OMP subtypes of Hib from patients with invasive disease and from carriers in the general population in The Gambia Source (no. of isolates)
No. of isolate in subtype: 2
4
5
8
9
NSAa
Blood (26) CSF (57) Pleural fluid (4) Synovial fluid (1) Pus (2) Total invasive (90)
4 3 0 0 0 7
10 34 3 0 2 49
1 5 0 1 0 7
2 5 0 0 0 7
2 2 0 0 0 4
7 8 1 0 0 16
Throat, noninvasive (64)
0
19
20
3
10
12
7
68
27
10
14
28
Total (154) a
NSA, no subtype assigned.
*_.
BIJLMER ET AL.
388
J. CLIN. MICROBIOL.
370
1000 24589
MW24589
9468-*Wp
a
"
........
_ o
W
20 subtypes (numbers at top) in nonheated specimens; 8 through 12, the same specimens modified by heat. On the vertical axis, right side, are shown the positions of the OMPs a to e; on the left side are shown molecular mass markers (in kilodaltons) and the position of the heat-modifiable OMPs a' and d' in unheated form.
Hib isolates were LPS serotyped for serotype 1. Only 1 of 90 invasive disease isolates were positive, as were 7 of 64 of the carrier strains from the general population. Epidemiology. OMP subtype 2 was isolated exclusively from patients with invasive disease (Table 2). OMP subtype S was isolated less frequently from invasive disease isolates than from healthy carriers in the general population (Fisher exact test, P < 0.001). A similar result was obtained for OMP subtype 9 (Fisher exact test, P < 0.05). OMP subtype 4 was found more frequently in invasive disease than in healthy throats (Fisher exact test, P < 0.01). The distribution of OMP subtype 8 and the nontypeable strains was similar for patients and healthy carriers. A significant correlation was found between L subtypes (subtype 2, 4, and 8) and invasiveness of Hib strains; non-L types (subtypes 5 and 9) were found more frequently among carriers (Fisher exact test, P < 0.001). The geographical distribution of OMP subtypes isolated from carriers in the general population shows clear differences (Table 3). OMP subtype 2 was not isolated from carriers in any of the three locations. Subtype 9 was not found in carriers living in Basse. In contrast, subtype S was present almost exclusively in this location. The distribution of the various subtypes was random over the respective
TABLE 3. Geographical distribution of OMP subtypes of Hib obtained from throat swabs in the general population of The Gambia (n = 64) OMP subtype
2 4 5 8 9 Nonsubtypeable Total
No. of isolates from following village:
Brikama/Lamin (west)
Brefet (center)
Basse (east)
0 4 2 1 3 5
0
1
0 12 18 2 0 6
11
38
15
3 0 0 7
locations (Fisher exact test, P = nonsignificant), except for subtypes 5 and 9 (Fisher exact test, P < 0.05).
DISCUSSION Distribution of H. influenzae sero- and subtypes in The Gambia. Serotype b is the predominant H. influenzae serotype causing invasive diseases in most parts of the world. In the United States (10), The Netherlands (7), Denmark and Norway (15), Sweden (22), and Finland (19), around 99% of H. influenzae invasive disease is caused by this serotype. In the population with a very high incidence of H. influenzae invasive disease, the Alaskan Eskimos, serotype b is also the most frequently isolated serotype (33). We found that sero-
type b accounted for 95% of H. influenzae isolates from meningitis patients in The Gambia, which is slightly lower than the 99% reported by us on a smaller series (5). Our observations correlate well with the incidence of 96.7% serotype b for H. influenzae from meningitis patients in neighboring Senegal. A surprisingly high proportion of serotype a isolates from patients with invasive disease has been reported from nonindustrialized areas (12, 16, 31). Serotype a hardly occurred among CSF isolates from The Gambia but accounted for 5.8% of all invasive strains (Table 1). Serotype a strains were also present in the throats of healthy children (unpublished data). A higher proportion of serotype a in The Gambia was reported by Wall et al., but the number of strains analyzed was small (31). We conclude that in West Africa, as elsewhere, serotype b is responsible for most of the invasive diseases caused by encapsulated H. influenzae. The distribution of biotypes did not differ significantly from that in other reports (15, 22, 28, 33). In invasive H. influenzae disease, biotype I (84%) and biotype 11 (13%)
predominated. We have found five distinct OMP subtypes among the Gambian Hib isolates. These subtypes were compared to the OMP subtypes as described by Barenkamp et al. and Van Alphen et al. (2, 3, 25, 28). Only subtype 2, identical to the Barenkamp subtype 2L, has been described previously. OMP subtypes 4, 5, 8, and 9 are new subtypes and have been designated as new numbers in the OMP subtyping system described by Van Alphen et al. (25, 28). We conclude that in The Gambia Hib has a pattern of OMP profiles different from those in other parts of the world. OMP subtypes and invasive disease. It has been reported that certain OMP subtypes cause different types of disease and that the distribution of subtypes among carrier strains differs from that of disease isolates (21, 24). L subtypes (subtypes 2, 4, and 8) were associated with invasive disease, whereas non-L subtypes (subtypes 5 and 9) were found more
MOLECULAR EPIDEMIOLOGY OF Hib IN THE GAMBIA
VOL. 30, 1992
often in healthy carriers (P < 0.001) (Tables 2 and 3). The predominance of subtype 5 among carrier strains may be explained by differences in the geographical distribution (Table 3). Subtype 9 strains probably have a low virulence, since they were significantly more frequent among carrier isolates than disease isolates in the catchment area. The significant difference in the geographical distribution of noninvasive Hib strains between the western part and the eastern part of the country is in contrast with data on subtype distribution in the United States (10). We conclude that the area of circulation of bacterial isolates apparently is limited, although the transmission rate of carrier strains in The Gambia was shown to be rapid (4). LPS serotyping has been developed relatively recently as an additional epidemiological tool for studying Hib disease. In Western Europe (Finland, Sweden, Norway, Denmark, The Netherlands, Germany, the United Kingdom, and France), with the notable exception of Iceland, LPS serotype 1 is the most prevalent type in invasive disease (25, 27). In the United States, the LPS serotype distribution among invasive isolates is much more diverse. In our study, LPS serotype 1 was found infrequently (4.2%) and seven of eight LPS 1 strains were noninvasive Hib. LPS serotype 1 is therefore probably not a virulence determinant for invasive disease. As we did not test for LPS serotypes other than LPS serotype 1, we cannot be certain that the Hib isolates of The Gambia do not possess a common LPS type. Implications of the epidemiological data for vaccination. This study has shown that invasive and noninvasive isolates of Hib in The Gambia differ in several important characteristics from isolates studied in other areas in the world. The data are important for the surveillance of Hib disease in developing countries and are baseline data for a Hib PRPconjugated vaccine trial in The Gambia. If, however, the currently available PRP-conjugated vaccines prove to be less efficacious than we expect them to be in countries with epidemiology similar to that in The Gambia, a set of representative OMPs from Hib, which this study has shown to be different from those found in the western world, should be included in an alternative Hib OMP vaccine (11, 18). ACKNOWLEDGMENTS Our thanks are due to Abdulie Sanneh for his indefatigable efforts in carrying out the field study and to Sam Appiah, Ignatius Baldeh, Ramu Njie, and Kutube Manneh for their contribution as laboratory technicians. This work was supported by grant W93-122 from the Foundation for the Advancement of Tropical Research, the Netherlands. REFERENCES 1.
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