0066-4804/09/$08.00
⫹0 doi:10.1128/AAC.00994-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Antibiotic Susceptibility and Characteristics of Neisseria meningitidis
Isolates from the African Meningitis Belt, 2000 to 2006: Phenotypic
and Genotypic Perspectives
䌤
Sara Thulin Hedberg,
1* Hans Fredlund,
1Pierre Nicolas,
2Dominique A. Caugant,
3Per Olce
´n,
1and Magnus Unemo
1National Reference Laboratory for Pathogenic Neisseria, Department of Laboratory Medicine, Microbiology, O
¨ rebro University Hospital,
O
¨ rebro, Sweden
1; French Forces Institute of Tropical Medicine (IMTSSA), WHO Collaborating Centre for Reference and
Research on Meningococci, Marseille, France
2; and WHO Collaborating Centre for Reference and
Research on Meningococci, Norwegian Institute of Public Health, Oslo, Norway
3Received 25 July 2008/Returned for modification 12 October 2008/Accepted 18 January 2009
Up-to-date information regarding the antibiotic susceptibility of Neisseria meningitidis strains from African
countries is highly limited. Our aim was to comprehensively describe the antibiotic susceptibilities of a
selection of N. meningitidis isolates recovered between 2000 and 2006 from 18 African countries, mainly those
within the meningitis belt. Susceptibilities to 11 antibiotics were determined using Etest for 137 N. meningitidis
isolates (stringently selected from 693 available isolates). The isolates were also characterized by serogrouping,
multilocus sequence typing, genosubtyping, and penA allele identification. All N. meningitidis isolates were
susceptible to ceftriaxone, chloramphenicol, and ciprofloxacin. No isolate produced
-lactamase. Only three
isolates (2%) displayed reduced susceptibility to penicillin G. The two isolates with the highest penicillin G
MICs were the only isolates showing reduced susceptibility to ampicillin and cefuroxime. One of these isolates
was also resistant to penicillin V. One percent of isolates displayed reduced susceptibility to rifampin, while
52% of the isolates were resistant to tetracycline, 74% were resistant to erythromycin, and 94% were resistant
to sulfadiazine. The MICs of rifampin and tetracycline seemed to be associated with the serogroup of the
isolates. In total, 18 sequence types (STs), 10 genosubtypes, and 8 different penA alleles were identified; the
most common were ST-7, P1.20,9,35-1, and penA4, respectively. A high level of correlation was found between
ST, genosubtype, and penA allele. In conclusion, N. meningitidis isolates from the African meningitis belt
remain highly susceptible to the antibiotics used. Regarding
-lactam antibiotics, rare isolates showed a
reduced susceptibility to penicillins, but the expanded-spectrum cephalosporins are not affected at present.
Invasive meningococcal disease is a significant health
problem worldwide. In Africa, particularly in the sub-Saharan,
so-called meningitis belt, epidemics of acute meningitis can
reach incidence rates of 1,000 cases per 100,000 inhabitants,
and in individual communities, attack rates as high as 1:10
for the population have been reported. During these
epi-demics, a mortality rate of about 10% is usually reported,
which most probably is an underestimation (14). These
epi-demics in the African meningitis belt have historically been
caused mostly by a limited number of Neisseria meningitidis
serogroup A clones. During recent years, strains of other
sero-groups, such as serogroups C, W-135, and X, have also been
involved (13).
Early antibiotic treatment of meningococcal disease is
cru-cial for keeping the case fatality rate and risk of sequelae as low
as possible. In Africa, the general recommendation for
treat-ment during endemic periods is (i) ceftriaxone in multiple
doses to also provide effective treatment of other presumptive
etiological agents of bacterial meningitis, such as Streptococcus
pneumoniae and Haemophilus influenzae; or (ii) multiple doses
of penicillins. During meningococcal epidemics, the
recom-mended treatment is (i) a single dose of chloramphenicol in oil
or (ii) a single dose of ceftriaxone (13, 30, 43). However,
increased levels of reduced susceptibility to penicillins have
been reported worldwide (22, 28, 32, 39, 42). This reduced
susceptibility has been due mainly to alterations in
penicillin-binding protein 2, encoded by the penA gene (3, 27, 32, 35).
Furthermore, though still rare, resistance to chloramphenicol
has been reported from Australia, France, and Vietnam (12,
25). This resistance is considered to be due mainly to the
presence of the catP gene, encoding the enzyme
chloramphen-icol acetyltransferase. Resistance to ceftriaxone has been
re-ported from India (18). However, these strains certainly need
to be examined further, comprehensively characterized
pheno-typically and genetically, and confirmed/disconfirmed by an
independent laboratory (20).
In the African meningitis belt, chemoprophylaxis of close
contacts of patients is rarely used and is not recommended by
the WHO (43). In the Western world, on the other hand,
chemoprophylaxis is often used, and ciprofloxacin and
ri-fampin are recommended antibiotics (30). However, in recent
years, there have been reports of reduced susceptibility to
ciprofloxacin (1, 6–8, 26, 31) as well as resistance to rifampin
from several countries worldwide (23, 29, 33).
Comprehensive data regarding the antibiotic susceptibility
of N. meningitidis in many African countries are limited, and no
* Corresponding author. Mailing address: Department of
Labora-tory Medicine, Microbiology, O
¨ rebro University Hospital, SE-701 85
O
¨ rebro, Sweden. Phone: 46 19 602 15 20. Fax: 46 19 12 74 16. E-mail:
sara.thulin-hedberg@orebroll.se.
䌤
Published ahead of print on 2 February 2009.
1561
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up-to-date extensive study of the antibiotic susceptibility of N.
meningitidis is at hand. Susceptibility testing, performed mainly
using the disc diffusion method and the most commonly used
antibiotics for treatment, is carried out mainly in hospital
lab-oratories in the meningitis belt. Disc diffusion methodology,
however, is hard to optimize, standardize, and quality assure,
which makes the results from different laboratories and
coun-tries difficult to compare.
The aims of the present study were to comprehensively
de-scribe the antibiotic susceptibilities (to 11 different antibiotics)
of invasive N. meningitidis isolates, strictly selected from 18
different African countries, mainly those within the meningitis
belt, during the period 2000–2006 and to genetically
charac-terize them.
MATERIALS AND METHODS
Bacterial isolates.The N. meningitidis isolates used for this study were from collections available at the WHO Collaborating Centers in Marseille, France, and Oslo, Norway, and from the National Reference Laboratory for Pathogenic Neisseria in O¨ rebro, Sweden. In total, between 2000 and 2006, 693 invasive isolates were recovered from patients, using routine bacteriological procedures, in 18 different African countries, mainly within the meningitis belt (Table 1). From this collection, all isolates with suspected reduced susceptibility according to initial testing, as well as isolates representing the prevalent phenotypes (se-rogroup and antibiogram) from each country, were included. When several isolates were related to the same epidemic outbreak, only one representative isolate was selected. One hundred thirty-seven invasive isolates were subse-quently examined, comprising serogroups A (n⫽ 82), W-135 (n ⫽ 38), X (n ⫽ 8), Y (n⫽ 7), and C (n ⫽ 1), with one nongroupable isolate (Table 1). For quality control, one serogroup A reference strain, OR173/87 (15, 19), was included. The reference strain was susceptible to all antibiotics tested, except for erythromycin (MIC⫽ 0.75 g/ml) and sulfadiazine (MIC ⬎ 256 g/ml).
Phenotypic antibiotic susceptibility testing.The MICs of penicillin G, ampi-cillin, penicillin V, ceftriaxone, cefuroxime, chloramphenicol, ciprofloxacin, ri-fampin, tetracycline, erythromycin, and sulfadiazine were determined for all isolates, using the Etest method (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar (Becton Dickinson and Company, Sparks, MD) supplemented with 5% sheep blood at 37°C in 5% CO2for 16 to 18 h (40). The present Etest
method-ology was previously evaluated in comparison to a reference method, i.e., agar dilution (41). The breakpoints used are shown in Table 2. The breakpoints used were from several different organizations, since no organization describes break-points for all examined antibiotics. All isolates were tested for-lactamase production by use of nitrocefin discs (AB Biodisk, Solna, Sweden).
Isolation of genomic DNA.Isolation of genomic DNA from the N. meningitidis isolates was performed using the MagNA Pure system (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. The DNA preparations were stored at 4°C prior to PCR.
MLST.Multilocus sequence typing (MLST) was performed mainly as previ-ously described (15, 17). The isolates were assigned a sequence type (ST) ac-cording to the Neisseria MLST website (http://pubmlst.org/neisseria/).
Genosubtyping.The porA gene was amplified by real-time PCR as previously described (19). The porA amplicons were purified using a Multiscreen PCR96
plate (Millipore, Bedford, MA) and vacuum filtration according to the manu-facturer’s instructions. The purified products were resolved in 50l of 10 mM Tris-HCl, and cycle sequencing PCR was performed as previously described (19). Subsequently, the products were purified using ethanol-sodium acetate precipi-tation and resuspended in 10l formamide (Applied Biosystems, Warrington, United Kingdom) according to the manufacturer’s instructions. The nucleotide sequences were determined using an ABI PRISM 3100 genetic analyzer (Ap-plied Biosystems, Foster City, CA). The deduced amino acid sequences of vari-able region 1 (VR1) and VR2 were assigned genosubtype numbers according to the Neisseria meningitidis PorA variable regions database (http://neisseria.org /perl/agdbnet/agdbnet.pl?file⫽poravr.xml), and the sequences of VR3 were as-signed numbers according to the PorA VR3 database (http://www.smprl.scot.nhs .uk/PorA_VR3.htm).
penA gene sequencing.The penA gene was amplified and sequenced as previ-ously described (4, 35). The isolates were assigned a penA allele number by using the N. meningitidis penA database (http://neisseria.org/perl/agdbnet/agdbnet .pl?file⫽penA.xml). Based on the different penA alleles, a phylogenetic tree was
constructed with TREECON (version 1.3b) software by using the Jin and Nei substitution model, the Kimura evolutionary model, an␣ value of 0.5, and the neighbor-joining method (38), as previously described (37).
RESULTS
Antibiotic susceptibility.
The results of antibiotic
suscepti-bility testing are summarized in Table 2. Briefly, all isolates
were
-lactamase negative and susceptible to ceftriaxone
(MIC
ⱕ 0.002 g/ml), chloramphenicol (MIC, 0.38 to 1.5 g/
ml), and ciprofloxacin (MIC, 0.002 to 0.012
g/ml). Three of
the isolates (2%) displayed reduced susceptibility to penicillin
G; two of these isolates were in serogroup A (isolated in
Ethiopia and Somalia), and one was in serogroup Y (isolated
in Senegal). The two isolates with the highest penicillin G
MICs (MIC
⫽ 0.25 g/ml and 0.38 g/ml for isolates in
sero-groups A and Y, respectively) were the sole isolates displaying
TABLE 1. Numbers and serogroups of N. meningitidis isolates
recovered in 18 different African countries, mainly those in
the meningitis belt, during the period 2000–2006, according
to year and country (n
⫽ 137)
bCountry Yr of
isolation
No. of isolates (serogroup distribution)a
Angola
2000
2 (A)
2004
1 (A)
Benin
2003
3 (2 A, 1 W-135)
2004
3 (1 A, 1 Y, 1 W-135)
2006
3 (2 W-135, 1 Y)
Burkina Faso
2001
5 (3 A, 2 W-135)
2002
2 (W-135)
2003
6 (3 A, 2 W-135, 1 Y)
2004
12 (7 A, 3 W-135, 1 Y, 1 NG)
Burundi
2002
2 (A)
Cameroon
2000
4 (A)
2001
5 (4 A, 1 W-135)
Central African
Republic
2001
2 (W-135)
Chad
2001
7 (6 A, 1 W-135)
2003
1 (W-135)
2004
1 (W-135)
2005
2 (W-135)
Djibouti
2001
1 (A)
Ethiopia
2000
1 (A)
2001
1 (A)
2002
2 (A)
2003
2 (A)
Ghana
2004
3 (2 A, 1 W-135)
Kenya
2005
2 (W-135)
Niger
2000
8 (A)
2002
5 (3 A, 1 X, 1 W-135)
2003
11 (4 A, 3 Y, 4 W-135)
2004
4 (3 A, 1 X)
2005
5 (3 X, 2 W-135)
Nigeria
2003
2 (1 A, 1 W-135)
2004
2 (1 A, 1 W-135)
Republic of the Congo
2001
4 (2 A, 1 C, 1 W-135)
Senegal
2000
2 (1 A, 1 W-135)
2001
3 (2 A, 1 W-135)
2002
2 (1 Y, 1 W-135)
Somalia
2002
2 (A)
Sudan
2000
6 (5 A, 1 W-135)
2001
6 (A)
Uganda
2006
2 (X)
a NG, nongroupable. bAll isolates were from cerebrospinal fluid.
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reduced susceptibility to ampicillin and cefuroxime. The
sero-group Y isolate was also resistant to penicillin V (MIC
⫽ 1.5
g/ml). Overall, 1 of 82 serogroup A isolates (⬍1%) displayed
reduced susceptibility to rifampin (MIC
⫽ 0.38 g/ml).
Fifty-two percent of the isolates were resistant to tetracycline, 74%
were resistant to erythromycin, and 94% were resistant to
sulfadiazine.
For rifampin, two susceptible populations could be
ob-served, one comprising mainly serogroup W-135 and Y
iso-lates, displaying very low MICs, and one with higher MICs,
comprising mainly serogroup A and X isolates (Fig. 1). For
tetracycline, a similar pattern was observed, with a resistant
population (MIC, 2 to 6
g/ml) of mainly serogroup A isolates,
belonging to ST-7 or ST-2859, and a susceptible population
(MIC, 0.064 to 0.38
g/ml) consisting of isolates representing
other serogroups and STs.
MLST, genosubtyping, and penA gene sequencing.
Eighteen
different STs (comprising five clonal complexes and six STs not
belonging to any clonal complex) were identified. ST-7 (47%),
ST-11 (20%), and ST-2881 (9%) were the most prevalent STs,
and clonal complex ST-5 (cc5) was the most common clonal
complex (58% of the isolates).
A total of 10 genosubtypes were identified, with P1.20,9,35-1
(58%), P1.5,2,36-2 (20%), and P1.5-1,2-36,36-2 (9%) being the
most prevalent.
Eight penA alleles were identified, with penA4 being the
most common (65%) (Fig. 2). penA4 was also found to be
associated with serogroup A and cc5 (Fig. 2). Accordingly,
TABLE 2. Susceptibilities of N. meningitidis isolates (n
⫽ 137) recovered during the period 2000–2006 in 18 different African countries,
mainly those within the meningitis belt, to 11 different antibiotics
Antibiotic Breakpoints
(susceptible/resistant)
MIC range
(g/ml) (MICg/ml)50 (MICg/ml)90
% of isolates
Susceptible Intermediate Resistant
Penicillin G
ⱕ0.094/⬎1
a0.006–0.38
0.047
0.094
98
2
0
Ampicillin
ⱕ0.12/⬎1
b0.016–0.75
0.064
0.094
99
1
0
Penicillin V
ⱕ1/⬎1
c0.016–1.5
0.19
0.38
99
0
1
Ceftriaxone
ⱕ0.12/
b⬍0.002–0.002
⬍0.002
⬍0.002
100
0
0
Cefuroxime
ⱕ0.25/⬎1
c⬍0.016–0.5
0.047
0.094
99
1
0
Chloramphenicol
ⱕ2/⬎4
b0.38–1.5
0.75
1
100
0
0
Ciprofloxacin
ⱕ0.03/⬎0.25
b0.002–0.012
0.004
0.004
100
0
0
Rifampin
ⱕ0.25/⬎1
b0.002–0.38
0.064
0.125
99
1
0
Tetracycline
ⱕ1/⬎1
d0.064–6
2
4
48
0
52
Erythromycin
ⱕ0.5/⬎0.5
d0.032–3
0.75
1
26
0
74
Sulfadiazine
eⱕ1/⬎4
b1.5–
⬎256
⬎256
⬎256
0
6
94
aBreakpoints previously described by Taha et al., based on a combination of identified penicillin G MICs and the presence/absence of a penA mosaic allele (32).
b
Breakpoints proposed by the European Meningococcal Disease Society (40).
c
Breakpoints in accordance with the Swedish Reference Group for Antibiotics (www.srga.org).
d
Breakpoints in accordance with the British Society for Antimicrobial Chemotherapy (4a).
e
Breakpoints for sulfisoxazole were used, which displayed a similar MIC distribution to that of sulfadiazine by Etest (data not shown).
FIG. 1. Serogroup distribution and MICs of rifampin for N. meningitidis isolates (n
⫽ 137) collected in 18 different African countries, mainly
those from the meningitis belt, from 2000 to 2006. The broken line indicates the breakpoint for susceptible isolates (MIC
ⱕ 0.25 g/ml).
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93% of all serogroup A and 97% of all cc5 isolates had the
penA4 allele.
The two isolates with the highest MICs of penicillin G
(MIC
⫽ 0.25 g/ml and 0.38 g/ml) comprised mosaic alleles
(penA52 and penA20, respectively), while all other isolates
displayed wild-type penA alleles. Consequently, there was an
association between reduced susceptibility to penicillins and
the presence of penA mosaic alleles in African meningitis belt
N. meningitidis isolates.
There was also an association between penA alleles and
genosubtypes (Fig. 2). Ninety-six percent of the isolates with
genosubtype P1.20,9,35-1 carried penA4, and 100% of the
ge-nosubtype P1.5,2,36-2 isolates contained penA1. Gege-nosubtypes
and STs were also associated, especially in studying the clonal
complexes. The most common genosubtype, P1.20,9,35-1 (n
⫽
79 isolates), consisted of only isolates belonging to cc5, and the
second most common genosubtype, P1.5,2,36-2 (n
⫽ 27),
con-sisted of only cc11 isolates.
DISCUSSION
In the present study, N. meningitidis isolates recovered in
2000 to 2006, mainly from countries constituting the African
meningitis belt, were found to remain highly susceptible to the
antibiotics used for treatment and prophylaxis. However,
iso-lates from northern and southern Africa were not included and
may differ substantially (10, 44).
Only a few isolates included in the study displayed reduced
susceptibility to penicillin G (2%) and other
-lactam
antibi-otics (1% had reduced susceptibility to ampicillin, penicillin V,
and cefuroxime). Since the study was based partly on isolates
selected for reduced antibiotic susceptibility, this proportion is
most likely lower among all clinical N. meningitidis isolates in
the African meningitis belt, where numerous isolates
repre-senting identical strains are recovered in outbreak situations.
All isolates were susceptible to ceftriaxone. In spite of the
fact that there have been reports of rare
chloramphenicol-resistant N. meningitidis isolates in Australia, France, and
Viet-nam (12, 25), all of the presently examined African
meningo-cocci were fully susceptible to chloramphenicol. Previous
studies of African N. meningitidis isolates have displayed
iden-tical results, i.e., all isolates were fully susceptible to
chloram-phenicol (11, 36, 44). Furthermore, all previously described
chloramphenicol-resistant isolates have been serogroup B
iso-lates (12, 25), which is extremely rare in Africa. Serogroup B N.
meningitidis strains are highly transformable, unlike serogroup
A strains (25), and hence the lower transformation rate for
serogroup A could be a possible explanation for the lack of
chloramphenicol resistance in the examined African N.
men-ingitidis isolates. However, serogroup A accounted for only
60% of the isolates in the present study, so this is not the only
explanation.
For continents other than Africa, there have been reports of
reduced susceptibility or resistance to ciprofloxacin (1, 6–8, 26,
31) and rifampin (23, 29, 33), which are commonly used
anti-biotics for prophylaxis. In the present study, however, the
ex-amined N. meningitidis isolates, mainly from the African
men-ingitis belt, were highly susceptible to both antibiotics, with
only one serogroup A isolate from Ethiopia displaying reduced
FIG. 2. Phylogenetic tree based on a 402-bp alignment of penA gene sequences in N. meningitidis isolates (n
⫽ 137) collected in 18 different
African countries, mainly those in the meningitis belt, from 2000 to 2006. The genosubtypes and clonal complexes (cc) associated with the different
penA alleles are included in the boxes.
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susceptibility to rifampin (MIC
⫽ 0.38 g/ml). This finding
may be due to the infrequent use of prophylaxis in Africa.
Tetracycline and erythromycin are not used for treatment of
meningococcal disease. Since these antibiotics are used for
treatment of many other infections, their susceptibility patterns
may reflect the overall antibiotic pressure on African N.
men-ingitidis isolates. A large number of the presently examined
African N. meningitidis isolates were resistant to tetracycline
(52%) and erythromycin (74%). A high level of resistance to
tetracycline (85%) has also been described in a previous study
including a limited number of African N. meningitidis isolates
(n
⫽ 20) (24). According to other previous reports (9, 16),
among 441 examined N. meningitidis isolates, including 22
se-rogroup A isolates, resistance to tetracycline was found only in
the serogroup A isolates, all belonging to cc5. This resistance
was associated with the drug efflux mechanism encoded by the
gene tet(B) (9). The close association between serogroup A
isolates belonging to ST-7 or ST-2859 and tetracycline
resis-tance observed both in the present study and in the previous
one (9) may explain the low resistance to tetracycline (0.3%)
observed in a previous French study including 2,167 N.
men-ingitidis isolates, with only nine serogroup A isolates (2). In
that study, 9.1% of all invasive isolates in France between
1999 and 2002 also displayed reduced susceptibility to
eryth-romycin (2).
In the present study, almost all examined African isolates
displayed wild-type penA alleles. The most common allele was
penA4, which was present in 65% of the isolates. This can be
compared to a recent extensive study where the penA genes of
N. meningitidis isolates (n
⫽ 1,670) from 22 mainly European
countries were sequenced (32). In this study, penA4 was found
in only 10% of the isolates, while penA1 and penA3 together
accounted for 46% of the isolates (32). Accordingly, during
recent years, i.e., 2000 to 2006, penA4 may seem strongly
as-sociated with cc5, and hence serogroup A (Fig. 2). This may be
due to the clonal expansion of cc5 in the meningitis belt since
the 1980s (5, 34). The two African isolates with the highest
MICs for the different penicillins displayed the penA mosaic
alleles 52 and 20. penA52 was found in a serogroup A, ST-7
isolate from Somalia. In the previous European study (32),
penA52 was found in 19 serogroup B isolates from six different
European countries and in 1 serogroup B isolate from the
United States. In the present study, penA20 was found in a
serogroup Y, ST-23 isolate cultured in Senegal. For
compari-son, in the previous European study (32), penA20 was found in
six serogroup Y isolates and one serogroup C isolate,
recov-ered in France and Italy.
In the present study, the most common ST was ST-7 (n
⫽ 64
isolates) (in cc5 [n
⫽ 79]), followed by ST-11 (n ⫽ 28) (in cc11
[n
⫽ 29]). These findings are in congruence with earlier studies
regarding N. meningitidis isolates from the meningitis belt (5,
15, 21, 34). In Africa, cc5 and cc11 have been predominant,
and only a few other sequence types, such as ST-2881 and
ST-181, have been detected sporadically (5). In the present
study, cc5 consisted of two subgroups of serogroup A isolates,
all of which were resistant to sulfadiazine, but 5 and
ST-2207 isolates were susceptible to tetracycline and ST-7 and
ST-2859 isolates were resistant. Both ST-7 and ST-2859 are
relatively new variants of cc5 and have recently expanded in
the meningitis belt to replace ST-5 (5). cc11 and ST-2881
consisted mainly of sulfadiazine-resistant,
tetracycline-suscep-tible serogroup W-135 isolates. In ST-181, only
sulfadiazine-and tetracycline-susceptible serogroup X isolates were found.
All of these isolates were cultured in Niger during 2002 to 2005
and probably represented a single clone, since all phenotypic
and genotypic characteristics for the five individual isolates
were identical, with the exception of slightly different MICs.
In conclusion, N. meningitidis isolates from the African
men-ingitis belt remain highly susceptible to the antibiotics used for
treatment and prophylaxis, and hence, the present results
sup-port the WHO recommendations (43). Regarding
-lactam
antibiotics, a few isolates displayed reduced susceptibility to
penicillins, but at present the expanded-spectrum
cephalospo-rins, such as ceftriaxone, are not affected. However, it is crucial
to continuously monitor the antibiotic susceptibility of N.
men-ingitidis to avoid future treatment failures in Africa during both
endemic and epidemic periods.
ACKNOWLEDGMENTS
This study was supported by grants from the O
¨ rebro County Council
Research Committee and the Foundation for Medical Research at
O
¨ rebro University Hospital, O
¨ rebro, Sweden.
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