International Circumpolar Surveillance Interlaboratory Quality
Control Program for Serotyping Haemophilus influenzae and
Serogrouping Neisseria meningitidis, 2005 to 2009
Raymond S. W. Tsang,aKaren Rudolph,bMarguerite Lovgren,cSadjia Bekal,dBrigitte Lefebvre,dLotte Lambertsen,eTammy Zulz,b
and Michael Bruceb
National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canadaa; Arctic Investigations Program, Centers for Disease Control and
Prevention, Anchorage, Alaska, USAb; Provincial Laboratory for Public Health, Edmonton, Alberta, Canadac; Laboratoire Santé Publique du Québec, Sainte-Anne-de-
Bellevue, Québec, Canadad; and Neisseria and Streptococcus Reference Laboratory, Statens Serum Institut, Copenhagen, Denmarke
diseases caused by Neisseria meningitidis and Haemophilus influenzae. An interlaboratory quality control (QC) program to
monitor laboratory proficiencies in the serogrouping of N. meningitidis and serotyping of H. influenzae strains was codeveloped
meningitidis and 26 H. influenzae isolates) were distributed among the five participating laboratories. The overall serogroup
concordance for N. meningitidis strains was 92.3% (96/104), without including three isolates that were found to express both
crepancies were observed most frequently for serogroups W135, X, Z, and 29E. The overall serotype concordance for H. influen-
cases, the serotypeable H. influenzae isolates were misidentified as being nontypeable. These data demonstrate a high degree of
concordance for serogroup and serotype determinations of N. meningitidis and H. influenzae isolates, respectively, among the
tially focused on the surveillance of invasive pneumococcal dis-
ease (IPD) in the U.S. Arctic (Alaska) and northern Canada and
began in 1999 (30). Soon thereafter, other countries joined
program has since expanded to cover the surveillance of other
invasive bacterial diseases caused by Neisseria meningitidis, Hae-
mophilus influenzae, and group A and B streptococci (29). The
surveillance of invasive bacterial diseases in the circumpolar re-
a ministerial forum of governments from countries in geograph-
ical proximity to the Arctic. The Invasive Bacterial Disease Work-
ing Group (IBDWG) within the ICS program is made up of both
laboratory scientists and clinical epidemiologists working in pop-
ulation/public health. The IBDWG has worked to form a labora-
collection for the long-term monitoring of invasive bacterial dis-
eases, with the ultimate aim of the prevention and control of in-
vasive bacterial diseases.
Essential for the success of any international surveillance
program is the need to be able to compare data, which are often
generated by using different testing methodologies. In 1999,
with the formation of the ICS program for the surveillance of
IPD, an interlaboratory quality control (QC) program was in-
troduced. This QC program served as an external proficiency
testing mechanism for the serotyping and antibiotic suscepti-
bility testing of Streptococcus pneumoniae and provided a
means for the monitoring of test results and standard testing
procedures across all participating laboratories (30). A similar
program that monitors laboratory proficiencies in the sero-
grouping of N. meningitidis and the serotyping of H. influenzae
isolates was codeveloped in 2004 by the Centers for Disease
Control and Prevention Arctic Investigation Program (AIP)
(Alaska) and the Public Health Agency of Canada National
Microbiology Laboratory (NML) (Winnipeg, Canada). The
other laboratories that participate in this QC program include
the Provincial Laboratory for Public Health (Edmonton, Al-
berta, Canada), Laboratoire Santé Publique du Québec
(Sainte-Anne-de-Bellevue, Québec, Canada), and Statens Se-
rum Institut (Copenhagen, Denmark).
The introduction of effective conjugated vaccines against sub-
populations of H. influenzae (serotype b) and N. meningitidis (se-
Received 13 July 2011 Returned for modification 23 September 2011
Accepted 7 December 2011
Published ahead of print 14 December 2011
Address correspondence to Raymond S. W. Tsang, raymond.tsang@phac
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0095-1137/12/$12.00Journal of Clinical Microbiologyp. 651–656jcm.asm.org
rogroups A, C, W135, and Y) has led to substantial changes in the
countries with childhood immunization programs for H. influen-
zae serotype b (Hib), invasive Hib disease has been largely con-
trolled, and the contention that Hib may be eliminated has also
been discussed (4). Subsequent to the introduction of Hib conju-
gate vaccines, non-serotype b H. influenzae has now become the
With the increasing use of vaccines to control invasive meningo-
coccal disease (IMD) caused by serogroups A, C, Y, and W-135
(12, 26) and the recent development and imminent licensure of a
serogroup B vaccine (33), a larger proportion of the remaining
IMD in the future will be caused by organisms with rare sero-
use, laboratory staff will have less experience in routine test pro-
cedures for the serogrouping and serotyping of these bacteria. At
the same time, the chances of encountering less common sero-
acteristics of the predominant organisms coupled with a lack of
adequate experience, the serotyping and serogrouping of bacteria
can become challenging tasks in the routine clinical microbiology
laboratory. Also, discrepancies in the identification of serogroups
of N. meningitidis and serotypes of H. influenzae have been re-
ported in the literature in the last decade (22, 27). Finally, newer
of these pathogens have been introduced in the last 2 decades (5,
7–9, 15, 18, 23, 25, 27, 32, 34, 36). All these developments point
toward the importance of the quality control of laboratory proce-
dures used to monitor these invasive bacterial pathogens, espe-
cially when vaccine usage and bacterial evolution are occurring in
Here we report the development of an interlaboratory quality
control program for the serogrouping of N. meningitidis and se-
years of this program (2005 to 2009).
MATERIALS AND METHODS
gitidis and H. influenzae isolates (5 each) were distributed among the
following laboratories two times per year (with the exception of 2008,
when the two panels were combined, with only one shipment of 10 iso-
lates): the Centers for Disease Control and Prevention Arctic Investiga-
tion Program (AIP), the Public Health Agency of Canada National Mi-
crobiology Laboratory (NML), the Provincial Laboratory for Public
Health (Edmonton, Alberta, Canada), the Laboratoire Santé Publique du
Québec (Sainte-Anne-de-Bellevue, Québec, Canada), and the Statens Se-
upon in advance. Each panel consisted of either two isolates of H. influ-
and three isolates of H. influenzae. H. influenzae isolates were selected to
represent either a known serotype or a nonencapsulated or nonserotype-
either a known serogroup or a nongroupable or nonserogroupable strain
(see Table 3). The NML distributed isolates on chocolate agar slants. The
AIP distributed lyophilized isolates prepared in skim milk. All isolates
were shipped according to International Air Transportation Association
Serotyping of H. influenzae. The most commonly used methods for
the serotyping of H. influenzae included either slide agglutination using
mel Europe Ltd., Dartford, United Kingdom) and PCR capsule typing
based on either a procedure described previously by Falla et al. (15) or a
multiplex PCR approach described previously by Gonin et al. (18). Some
laboratories employed more than one approach for serotype identifica-
tion (Table 1).
Serogrouping of N. meningitidis. For N. meningitidis, the identifica-
tion of the serogroup was accomplished by slide agglutination using in-
mercial latex particle agglutination test kits for serogroups A, B, and C
(Slidex [bioMérieux Clinical Diagnostics, France], Pastorex meningitidis
kit [Bio-Rad Laboratories], and Wellcogen [Remel]) were used together
sera for serogroups W135, X, Y, Z, and 29E (2). Other methods included
PCR for the identification of serogroups A, B, C, Y, W135, X, Z, and 29E
TABLE 1 Methods and reagents used for serotyping of Haemophilus influenza and serogrouping of Neisseria meningitidis
Serotyping of H. influenzaeSerogrouping of N. meningitidis
Method Source, reagent, or reference(s)MethodSource, reagent, and/or reference(s)
1Slide agglutinationDifco LaboratoriesReal-time PCRa
2 Slide agglutinationRemel (Thermo Fisher)Slide agglutination
Antisera provided by the National Microbiology Laboratory;
8, 9, 19, 36
3PCR15, 18Slide agglutinationAntisera provided by the National Microbiology Laboratory
4 Slide agglutinationDifco LaboratoriesLatex agglutinationSlidex (Pasteur-Merieux), Wellcogen (Remel), Pastorex
meningitis kit (Bio-Rad Laboratories)
In-house group-specific rabbit antisera; 2
8, 9, 36
In-house-produced rabbit antisera; 3
5, 8, 9, 36
aPCR targeting the sacB gene of serogroup A, the siaD genes of serogroups B and C, the synF gene of serogroup Y, the synG gene of serogroup W135, and the xcbB gene of
bPCR targeting the sacB and mynC genes of serogroup A, the siaD genes of serogroups B and C, the synF gene of serogroup Y, and the synG gene of serogroup W135.
cPCR targeting the sacB and mynC genes of serogroup A, the siaD genes of serogroups B and C, the synF gene of serogroup Y, and the synG gene of serogroup W135.
dPCR targeting the sacB gene of serogroup A, the siaD genes of serogroups B and C, the synF gene of serogroup Y, the synG gene of serogroup W135, and unique regions of the ctrA
genes specific for serogroups 29E, X, and Z.
Tsang et al.
jcm.asm.orgJournal of Clinical Microbiology
one method to determine the serogroup nature of N. meningitidis strains
Reporting. A standardized report form to ensure consistent data col-
lection was sent by the distributing laboratory with each QC panel. The
information collected on the report form included the test method used
and a table for reporting each serotype/serogroup. The report form was
completed by each participating laboratory and then returned to the dis-
tributing laboratory within 6 weeks. The distributing laboratory was re-
sponsible for compiling a summary report of the results, and if specific
problems were identified, a discussion was offered by the distributing
laboratory to assist with problem solving.
the province, state, or country from which they were distributed. The
isolates could be retained for internal reference use but were not shared
with other laboratories or used for research purposes by the receiving
laboratories without written consent from the distributing laboratory.
Isolates distributed for quality control. During the first 5 years
isms (26 H. influenzae and 24 N. meningitidis isolates) were dis-
tributed among the five participating laboratories. The serotype
and serogroup characteristics of the QC organisms are described
the developed protocol were as follows: (i) in 2008, all 10 QC
organisms were sent out in one panel instead of two due to some
logistic difficulties, and (ii) one laboratory did not participate in
the testing of the second panel in 2006.
Serotyping of H. influenzae. Of the 127 attempts carried out
for an overall concordance of 98% (125/127 attempts). Five sero-
type a and five serotype b isolates were correctly identified by all
laboratories throughout the 5 years of this program. Concordant
serotyping results were also obtained among all laboratories for
four isolates of serotype f, 4 isolates that were nontypeable, 2 iso-
e. Discrepancies between the expected serotype and the reported
results were noted in two instances (Table 2). The two discrepant
results involved a serotype c strain and a serotype e strain distrib-
uted in two separate panels. In both cases, the serotypeable H.
influenzae strains were identified as being nontypeable; in one
laboratory, this was done by a bacterial agglutination test, and in
the other laboratory, this was determined by multiplex PCR.
isolates were distributed to the five participating laboratories for
serogroup determinations. Three isolates were subsequently
found to express both serogroup Y and W135 specificities and
were excluded from the analysis to obtain the percent concor-
fore, of the 104 attempts to serogroup these 21 N. meningitidis
dance of 92.3% (96/104 attempts) (Table 3). Concordant results
were obtained among all laboratories for the identification of se-
rogroups A (1 isolate), B (5 isolates), C (3 isolates), and Y (2
and 29E as well as for the 3 unusual isolates that expressed both
serogroup Y and W135 antigenic specificities. The discrepancies
Three laboratories reported incorrect results for two serogroup Z
and two serogroup 29E organisms. One laboratory reported in-
isms. This laboratory was using a PCR protocol that allowed the
cause the slide agglutination result was weak for serogroups A, B,
not allow for the identification of serogroup Z isolates. Finally,
W135, X, and Y, reported a serogroup 29E organism as being a
serogroup Y organism, which could not be explained. Depending
on the methods used, laboratories reported the three unusual
either serogroup Y or serogroup W135 strains (Table 4).
After 5 years of this QC program, our results indicate a high con-
ing of N. meningitidis (92%) isolates among the participating
TABLE 2 Serotype results for the H. influenzae quality control isolates
of the International Circumpolar Surveillance program, 2005 to 2009
(no. of isolates)b
% concordance (no. of
total no. of tests)
a, b, c, e, f
a, b, c, d, e, f
a, b, d, NT (2)
a, b, e, f, NT
a, b, e, f, NT
aA serotype c isolate and a serotype e isolate were reported as being nontypeable.
TABLE 3 Serogroup results for the N. meningitidis quality control
isolates of the International Circumpolar Surveillance program,
2005 to 2009
Serogroups (no. of
% concordance (no. of
total no. of tests)a
A, B, C, Y, W135
B, W135, X, Y/W135
C, X, Z (2), 29E
B (2), W135, Y/W135 (2)
B, C, Y, W135, 29E
aThree isolates appeared to express antigenic specificities of both serogroups Y and
W135 and were excluded for the calculation of concordance.
bOne laboratory did not participate in the autumn panel.
cInvolved one laboratory that identified a serogroup W135 organism as a serogroup X
organism and that identified a serogroup X organism as a W135 organism, possibly due
to a mix-up of the specimens.
dOne laboratory failed to identify the serogroup Z organism twice (spring and autumn
panels), and another laboratory failed to identify it once; both laboratories were
employing the PCR method and reported these organisms as being nongroupable.
Another laboratory using real-time PCR misidentified a serogroup 29E organism as a
serogroup Y organism. One laboratory, also using PCR, did not identify the serogroup
29E strain and reported it as being nongroupable.
eOne laboratory using PCR identified the serogroup 29E organism as being
Quality Control for H. influenzae and N. meningitidis
March 2012 Volume 50 Number 3jcm.asm.org 653
tivity of the slide agglutination test (where the reader determines
agglutination visually) and the use of PCR by some laboratories.
The only discrepancies for H. influenzae were detected in the
first year of the program, and since then, no errors have been
found. In one case, a serotype e isolate was misidentified as being
nontypeable by the multiplex PCR used (18). Investigation into
that case indicated that the multiplex PCR failed to detect the
capsule-specific primers with primers that target the 16S gene for
the identification of H. influenzae. Individual PCR setups using
the different sets of primers separately showed the presence of the
bexA gene as well as the serotype e gene in the QC isolate. Subse-
quently, this laboratory changed their PCR protocol for the iden-
tification of H. influenzae serotypes from a multiplex PCR to a
PCR procedure that uses the different sets of primers separately
instead of mixing them up in one reaction. In the other case, the
serotype c isolate was misidentified by a slide bacterial agglutina-
tion test. Upon the repetition of the test, a positive identification
was obtained. Therefore, the actual reason for the original false-
negative result was not obvious but might be related to the rare
occasions of encountering a less common serotype of H. influ-
Serogroup determinations for the five most common sero-
also accurate for the most part among the participating laborato-
ries, with only one laboratory reporting an error for a serogroup
Most of the discordant results involved either rare serogroups
antigenic specificities. The inability to detect the rare serogroups
(serogroups X, Z, and 29E) was due mainly to either not using
test or using PCR protocols that did not include primers to detect
them. Many current PCR protocols for the detection and identi-
fication of N. meningitidis serogroups, including those used by
meningitidis in this study, use primers that target either the ctrA
gene alone or in combination with primers that detect the sacB
(35) or mynC (19) gene for serogroup A, the siaD genes for sero-
W135, respectively (8, 9, 36). As such, serogroups such as sero-
groups X, Z, and 29E will not be identified by these protocols.
that uses primers designed to bind to the unique regions of the
ctrA genes of strains of serogroups X, Z, and 29E (5).
Our current finding of difficulties in the identification of rare
serogroups is in agreement with the results of a previous compar-
ative study of three commercial diagnostic tests for the identifica-
67 to 88% (40). The uncommon serogroups W135, X, Y, Z, and
29E were mainly responsible for the poor performance. Although
serogroups X, Z, and 29E are rare causes of invasive meningococ-
cal disease, they have been reported to cause disease (16, 41) or
even outbreaks (17). The detection and identification of the rela-
tively rare serogroups X, Z, and 29E based on a PCR assay that
targets the sequence variations at the 5= ends of their ctrA genes
have been described (5). With common serogroups (serogroups
A, C, Y, and W135) being controlled by vaccination, rare sero-
groups may become relatively more common causes of IMD than
before, and therefore, laboratories may wish to adopt and update
their laboratory protocols to be able to detect these unusual or-
Recently, we (31, 39) as well as others (13) have described a
small percentage (1.3%) of serogroup Y and serogroup W135 N.
meningitidis strains expressing capsules with both serogroup Y
and W135 antigenic specificities. Serogroup Y and W135 menin-
gococci have related capsular structures (6), and both are het-
eropolymers of either glucose linked to sialic acid (for serogroup
ber of PCR assays for the differentiation of serogroups Y and
W135 have been described (9, 27, 36), but none is based on the
the enzymes responsible for the final capsule structures. As a re-
were classified as serogroup Y strains by PCR (27), but they may
show either agglutination in anti-W135 antiserum or agglutina-
identification of Y-W135 isolates requires the detection of the
The serogrouping or serotyping of the capsular antigens of H.
influenzae and N. meningitidis by the slide agglutination test is
known to be subjective, technically demanding in skill and expe-
rience of the laboratory personnel, and dependent on the quality
of the antisera. As a result, discrepancies in the serotyping results
for H. influenzae and serogrouping results for N. meningitidis
strains have been reported in the literature. For example, in an
Active Bacterial Core (ABC) surveillance study that compared re-
sults of slide agglutination tests performed at state health depart-
for Disease Control and Prevention, 56 of 141 (40%) invasive H.
influenzae isolates collected between January 1998 and December
TABLE 4 Serogrouping results from the five participating laboratories in the International Circumpolar Surveillance program for the three unusual
N. meningitidis isolates expressing both serogroup Y and W135 antigenic specificities
Serogroup(s) determined (method[s]) by laboratorya:
2006-07W135 (PCR) W135 (bacterial
W135 (bacterial agglutination) Did not analyzeW135 (bacterial agglutination),
W135 (bacterial agglutination),
W135 (bacterial agglutination),
2008-09 Y (PCR)Y (bacterial agglutination)W135 (bacterial agglutination)
2008-10Y (PCR) NG (bacterial agglutination),
W135 (bacterial agglutination and PCR)
Tsang et al.
jcm.asm.orgJournal of Clinical Microbiology
1999 were found to give discrepant results (23). In another study
gia Emerging Infections Program from 1 January 1989 to 31 De-
when slide agglutination test results were compared to results ob-
tained by PCR capsule typing (34). In a study in Brazil, slide ag-
glutination results obtained either by first screening with an anti-
serotype b antiserum followed by testing with antisera against
individual serotypes (anti-serotype a, c, d, e, and f antisera) if
1) or by testing individual isolates with antisera against all six
serotypes (anti-serotype a, b, c, d, e, and f antisera) (method 2)
were compared to results obtained by capsule typing by PCR (7).
In that study, the agreement rate between slide agglutination
method 1 or 2 and PCR for invasive isolates was 68% or 88%,
between slide agglutination results and PCR typing were 46.5%
and 94.2% for methods 1 and 2, respectively (7). For N. meningi-
tidis, 58 of 1,298 isolates, or 4.5% of isolates, gave discordant se-
rogrouping results when the results of the slide agglutination test
were compared with those of a real-time PCR method (27).
Another source that can contribute to discrepancies lies in the
methods used to determine serotyping and serogrouping results.
On one hand, the bacterial agglutination test is subjective to read
and prone to false-positive results due to the potential cross-
reactivities of antisera and is also less sensitive than newer molec-
do not detect the actual capsule but detect only the genes that
true absence of capsules, but their genes may still be detectable by
molecular approaches. This scenario can lead to true discrepan-
for any particular laboratory. An example of this type of discrep-
ancy is the detection of Hib-minus strains, as they carry the gene
this strain, both phenotypic and molecular techniques are re-
quired, and therefore, quality assurance programs like the one
described in this report are essential for surveillance studies to
monitor changes in the bacterial population.
Although various methods, such as latex agglutination (25),
rent immunoelectrophoresis (20), and monoclonal antibodies
with enzyme-linked immunosorbent assays (ELISAs) (37), have
been proposed to improve the determination of the serotype and
serogroup antigens, none has gained wide acceptance, possibly
because either reliable reagents are not available commercially or
the procedure is tedious or requires large volumes of antisera.
and reagents have proven that the slide agglutination method can
still be a very useful and simple tool for the identification of sero-
ized reagents and methods, quality control and external profi-
ciency programs are also essential for ensuring the accuracy of
testing. Because commercial vendors do not offer external profi-
ciency testing programs for these specialized assays, the ICS Inva-
sive Bacterial Disease Workgroup (IBDWG) incorporated exter-
nal proficiency testing into the ICS program to ensure the
accuracy of data collection for the surveillance of S. pneumoniae
(30) as well as of H. influenzae and N. meningitidis.
This QC program has heightened the participating laborato-
ries’ awareness of rare N. meningitidis serogroups and led to the
identification of three unusual N. meningitidis strains that might
not have been detected (13, 31, 39). As these vaccine-preventable
bacterial disease agents continue to evolve either as a result of
vaccine pressure or as a response to the natural immunity devel-
of these invasive bacterial agents. Besides the QC of serotyping of
H. influenzae and serogrouping of N. meningitidis, the IBDWG
will also consider in the future the addition of antibiotic suscepti-
bility testing and maybe nucleic acid testing (i.e., nonculture or
meningitidis from clinical specimens) to the current proficiency
We thank staff in the participating laboratories for their technical assis-
tance and support of this proficiency program.
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