Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Neisseria meningitidis PorA variable regions: rapid

detection of P17 and P119 variants by PCR
I. de Filippis1, C.F. de Andrade1, A.E.C.C. de Almeida1, M.M. Clementino1, C.A.C.M. Fernandes1,
M.L. de Carvalho2 and A.C.P. Vicente3
1 Microbiology Dept., National Institute for Quality Control of Health, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil
2 Secretaria de Vigilancia em Saude MS, Brasilia, Brazil
3 Genetic Dept., Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil

Abstract

Keywords
Neisseria meningitidis, PorA, serosubtypes,
typing methods, VR1 variants.
Correspondence
Ivano de Filippis, Microbiology Dept., National
Institute for Quality Control of Health-INCQS,
Oswaldo Cruz Foundation-FIOCRUZ, Rio de
Janeiro, Brazil, 21045-900.
E-mail: ivano.defilippis @incqs.fiocruz.br

2006 0795: received 1 June 2006, revised

and accepted 31 May 2007
doi:10.1111/j.1472-765X.2007.02205.x

Aim: Rapid characterization of variable region (VR)1 variants of the porA gene
among invasive strains is crucial for outbreak management and epidemiology
studies. Recent sequence analysis studies in Brazil showed that the VR1 P17
and P119 variants are highly prevalent, accounting for 68%, of the total number of VR1 variants characterized. The aim of this work is to develop a rapid
polymerase chain reaction (PCR)-based method for genosubtyping Neisseria
meningitidis by detection of porA variable regions P17 and P119.
Methods and Results: PCR primers for the detection of porA VR1 P17 and
P119 were designed and tested using 198 clinical N. meningitidis isolates that
had been previously evaluated by porA sequencing. All 50 strains with VR1
P17 and all 65 strains with VR1 P119 were positively identified by the respective VR-specific PCR and no false-positive reactions occurred.
Conclusions: VR-specific PCR amplification accurately identified VR P17 and
P119 strains.
Significance and Impact of the Study: To overcome the disadvantages of serosubtyping and sequencing for typing the porA VR1 segment of N. meningitidis,
we developed a PCR-based method to rapidly and accurately detect VR1 P17
and P119 variants. This approach is highly specific and sensitive; moreover it
may allow for genotype determination of culture-negative samples.

Introduction
Neisseria meningitidis is a major cause of meningitis and
septicaemia in children and adults throughout the world
(Achtman 1995). Characterization of meningococcal
strains isolated during outbreaks is crucial in understanding the epidemics and requires specific typing of isolates.
At least 13 serogroups have been defined by the serological specificity of the bacterial polysaccharide capsule
(Peltola 1983) however only serogroups A, B, C, Y and
W-135 isolates are major causes (about 90%) of
meningococcal meningitis and septicaemia. Other
important structures widely studied in meningococci are
the outer membrane proteins (OMP), subcapsular antigens the variation of which forms the basis for strain
426

characterization by serotyping and serosubtyping (Frasch

et al. 1985). These porin proteins of Neisseria meningitidis
are important components of OMP-based vaccines.
All meningococci possess class 2 or class 3 OMP called
PorB and virtually all strains expose on their outer membrane surface a class 1 porin protein called PorA (Tsai
et al. 1981). The secondary structure of class 1 OMP
shows eight extramembranous surface-exposed loops
designated IVIII (van der Ley et al. 1991). Two of these
loops contain variable regions, named VR1 (loop I) and
VR2 (loop IV), with most amino acid variation detected
within these regions. Amino acid sequences found among
VR are stable within an isolate but vary between unrelated isolates, and thus have been used for differentiation
of meningococci in a subtyping system. For this reason,

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I. de Filippis et al.

PorA is an important epidemiological marker in meningococcal disease and has been used as an immunogenic
antigen in several nonpolysaccharide-based serogroup B
meningococcal vaccine trials (Claassen et al. 1996;
Jessouroun et al. 2004; Sexton et al. 2004; Vermont et al.
2004; Devoy et al. 2005). The nomenclature so far used
to indicate PorA VR epitopes is P17-1,16-1, where P1
indicates the class 1 PorA protein, 7-1 and 16-1 are the
VR1 and VR2 families and their variants designated by
consecutive numbers. Neisseria meningitidis isolates have
been conventionally typed into serosubtypes by monoclonal antibodies (Mab) against PorA VR epitopes
(Abdillahi and Poolman 1988). However, many of these
VR are not recognized by existing Mab panels and indeed
the provision of a comprehensive panel would be impractical (Jolley et al. 2007). Thus, the serosubtype system is
incomplete, and at least 50% of the isolates are only
partially subtyped or not subtyped at all. This has been
explained by the presence of epitopes to which Mab are
unavailable (Suker et al. 1994), masking of epitopes on
the cell surface (Wedege et al. 1993) or lack of expressed
PorA in some individual isolates (Arhin et al. 1997).
Polymorphism analysis of the porA gene by nucleotide
sequencing (PorA VR-typing) has been used to overcome
the flaws presented by the serosubtyping method, predicting VR amino acid sequences (Suker et al. 1994; Feavers
et al. 1996; Jolley et al. 2007). During the last decade,
diagnostic and typing molecular methods using genomic
DNA sequencing became more accurate, cheaper and
faster, hence many public laboratories in developed countries, introduced such methods in their routine analysis of
infectious diseases. However, for developing countries,
these methods are still too expensive to be established in
their routine. Nevertheless, polymerase chain reaction
(PCR)-based methods have been widely used in developing countries in order to characterize many infectious
diseases aetiological agents.
A large database containing VR1 and VR2 sequences of
thousand strains isolated around the world can be found
at http://neisseria.org/nm/. According to this database, the
amino acid sequence variation found among VR1 and
VR2 is considerably high, e.g. more than 135 unique peptide sequences have been reported for VR1, grouped into
10 families. Worlwide, the incidence of P17 and P119
variants together according to the meningococcal MLST
(Multilocus Sequence Typing) database, excluding the
nontypeable strains, is 307% (22% and 87%, respectively) meanwhile the most incident VR1 variant P15,
accounts for 349% of all the typed strains. This is supported by several studies reporting that the prevalence of
P17 and P119 epitopes around the world, has been
found to be 316% (Molling et al. 2000; Clarke et al.
2001, 2003; Russell et al. 2004; Urwin et al. 2004). It is

Rapid detection of N. Meningitidis PorA VRs

important to emphasize that more than 75% of the

strains expressing any VR1 P17 or P119 variant, belong
to one of the seven hypervirulent clonal complexes which
have been implicated in severe epidemics worldwide.
These two PorA epitopes have been extensively used in
several meningococcal outer membrane vesicle (OMV)
vaccine preparations to improve immunogenicity of polysaccharide vaccines against serogroup B meningococci
(Feavers et al. 1996; Cartwright et al. 1999; Diggle and
Clarke 2003; Urwin et al. 2004).
In Brazil, PorA variants P17 and P119 are widely distributed among strains causing invasive meningococcal
disease. In a previous study (de Filippis and Vicente
2005), we showed that these epitopes are present in 68%
of the strains isolated from four states in Brazil located in
the most populated regions of the country (northeast,
southeast and south). Other studies on PorA types in
Brazil also showed a high incidence of these two genotypes (Sacchi et al. 2001). Based on this information, we
developed a PCR-based method to type N. meningitidis in
terms of their PorA VR1. The method described here
allowed the characterization of VR1, P17 and VR1, P119
families described in the MLST database.
Materials and methods
The porA gene in the N. meningitidis MC58 genome
(GenBank Acession Number AE002098) is an 1179-bp
sequence containing several variable regions of different
lengths, scattered along the gene extension. According to
the N. meningitidis PorA variable regions database, VR1
expresses 11 different families with 135 variants with a
mean length of 60 nucleotides (nt) for each variant. It also
describes 26 and 28 variants for P17 and P119 families,
respectively. Variant numbers are designated according to
variation within the amino acid sequences of VR1 families.
Every single amino acid change within the sequence produces a new variant. After alignment of all the P17 and
P119 variants deposited in the PorA database, we
observed that VR1 P17 and P119 variants also display
variable and nonvariable regions, within their original
sequences. This feature allowed the design of primers specific for the nonvariable regions of each epitope for the
amplification of the specific families P17 (P17-F: 5-GCA
CAA GCC GCT AAC GGT G-3) and P119 (P119-F:
5-CCG CCC TCA AAG AGT CAA CCT C-3) within VR1
and their variants. Each specific primer was used in the
forward direction with a reverse primer (Common-R:
5-GGCATTAATTTGAGTGTAG-3) specific for a common sequence found in position 11311149 of the
N. meningitidis MC58 porA gene (Fig. 1). The sequence,
from which the Common primer was generated, was found
in 56 porA gene sequences available in GenBank.

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427

Rapid detection of N. Meningitidis PorA VRs

I. de Filippis et al.

Figure 1 Schematic representation of the

porA gene of Neisseria meningitidis MC58
showing the localization of P17, P119 and
Common primers within variable region
(VR)1.

We analysed 198 N. meningitidis strains isolated from

the cerebrospinal fluid (CSF) of patients with meningococcal meningitis. The porA VR1 of the strains were
determined by sequencing prior to PCR analysis. Three
reference strains were also analysed, representing the two
target epitopes (ATCC 700110 and 700112 = P17 and
ATCC 700111 = P119).
The isolates were cultured on chocolate agar for 18
20 h at 37C in 5% CO2. Serogroups were determined by
latex agglutination and species confirmation was performed by nspA-PCR as previously described (de Filippis
et al. 2005). The strains analysed in the present study,
were preserved by freeze drying, and were part of the
INCQS Culture Collection of the Department of Microbiology, National Institute for Quality Control of Health,
FIOCRUZ, Rio de Janeiro, Brazil.
After growth in GC chocolate agar (BD, Franklin Lakes,
NJ, USA) at 37C in a 5% CO2 atmosphere for 24 h, cells
were picked from agar plates and DNA was extracted and
purified using the Dneasy Tissue Kit (Qiagen, Hilden,
Germany), according to the manufacturers instructions.
Purified DNA was stored at 4C prior to PCR.
Fifty-four CSF samples previously confirmed as positive
for meningococcal DNA by nspA-PCR were also analysed.
Because of the difficulty in performing serosubtyping analysis on CSF, we did not have access to the serology
results to compare with our PCR-based method. However, we sequenced the porA gene on those samples, in
order to confirm our PCR results (data not shown).
PCR reaction was performed using the three primers
previously described. Briefly, each reaction was performed
in a final volume of 50 ll using 1 U Taq DNA polymerase (5 U ll)1) (Promega, Madison, WI, USA) 1X
PCR buffer, 1 ll of deoxynucleotide triphosphate mixture (2 mmol l)1) (Invitrogen, Carlsbad, CA, USA),
2 mmol l)1 of MgCl2, 1 ll of each primer (5 pmol ml)1)
and 1 ll of DNA template. The amplification cycles were
94C for 10 min, followed by 30 cycles of 95C for
1 min, 40C for 1 min and 72C for 2 min and one cycle
of 72C for 5 min. PCR products were resolved by 1%
agarose gel electrophoresis at 100 V for 30 min in 05X
Tris-borate-EDTA (pH 80) buffer with a 100 bp molecular size marker (Invitrogen). The gels were stained with
ethidium bromide, and the gel images were digitalized
with the Video Documentation System and analysed with
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ImageMaster software (Amersham Pharmacia Biotech,

Uppsala, Sweden).
Results
The combination of primers P17-F + Common-R and
primers P119-F + Common-R, produced 1023-bp and
1008-bp amplicons, corresponding to VR1 variants P17
and P119, respectively (Fig. 2). No amplification
occurred when strains expressing VR1 variants different
from P17 and P119 were tested (Table 1). In addition,
cross amplification of both VR was not observed when

Figure 2 Polymerase chain reaction (PCR) amplification of variable

region (VR)1 P17 and P119. MW: 100-bp molecular size marker (Invitrogen); lane 1: Neisseria meningitidis ATCC 700110 (B:15:P17,1);
lane 2: N. meningitidis ATCC 700112 (B:4:P17,16); lane 3: N. meningitidis clinical strain (B:NT:P17,16); lane 4: N. meningitidis clinical
strain (B:NT:P17-1,1); lane 5: N. meningitidis clinical strain (B:NT:P172,3); lane 6: N. meningitidis clinical strain (B:NT:P17-4,1); lane 7:
N. meningitidis ATCC 700111 (B:4:P119,15); lane 8: N. meningitidis
clinical strain (B:NT:P119,15); lane 9: N. meningitidis clinical strain
(B:NT:P119-18,1); lane 10: N. meningitidis clinical strain (B:NT:P11911,15); lane N: negative control.

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I. de Filippis et al.

Rapid detection of N. Meningitidis PorA VRs

Table 1 Amplification of variable region (VR)1 variants of porA

PCR amplification*
Strain classification

No. of strains tested

P17-F

P119-F

B:P17
B:P17-4
B:P17-1
B:P17-2
C:P17-2
B:P17-9
B:P17-23
B:P17-13
B:P119
B:P119-18
B:P119-1
B:P119-11
B:P119-27
B:P11
B:P12
B:P13
C:P15
B:P15-1
C:P15-1
C:NT:P15-2
B:P15-8
B:P19
B:P112
B:P112-5
B:P114
B:P114-6
B:P114-8
B:P115
B:P116
B:P117-1
C:P118-1
B:P118
B:P120
B:P121-6
B:P121-13
B:P122
B:P122-1
Total

32
2
10
2
1
1
1
1
60
2
1
1
1
1
3
2
8
2
3
2
1
10
1
21
4
1
1
1
1
1
2
1
3
1
1
7
5
198

+
+
+
+
+
+
+
+

+
+
+
+
+

Primers P17-F and P119-F were used with Common-R primer to

amplify P17 and P119 VR1 regions.
PCR, polymerase chain reaction.

attemping to amplify P17 subtypes with P119-F +

Common-R and P119 subtypes with P17-F + CommonR. The two amplicons, with 1023 bp and 1008 bp, were
sequenced in order to confirm the DNA-amplified fragments (data not shown).
All the strains and CSF analysed, previously genotyped
through sequencing of the porA gene, were typed by PCR.
Of the 198 N. meningitidis strains analysed, 50 belonged
to P17 families with the following variants (7-1, 7-2, 7-4,
7-9, 7-13, 7-23) and 65 belonged to P119 with the following variants (19-18, 19-1, 19-11, 19-27) (Table 1).

From the 54 CSF samples genotyped by PCR, 15 were

positive for P119 and 10 for P17, which was confirmed
after sequencing of the porA gene (data not shown).
Discussion
Rapid diagnosis of invasive meningococcal disease is vital
to help physicians in patients management. The first prophylactic measures after the confirmation of meningococcal disease, are antibiotic administration to primary and
secondary contacts. When an increase in the number of
cases within a population limited to a geographic area is
detected, health authorities must rapidly determine the
circulating serogroups and serosubtypes to introduce
proper containment measures such as vaccination.
Meningococcal serogroups are easily characterized by
serology methods using commercial polyclonal antibodies
against the most important serogroups causing disease
(A, B, C, W135, Y), directly from CSF or from isolates.
However, serotyping and subtyping are conventionally
determined by the enzyme-linked immunosorbent assay
(ELISA), using Mab against epitopes of the class II or III
PorB protein and class I PorA protein, respectively. Characterization of meningococcal serotypes and subtypes by
this method requires multiple assays also; this methodology is generally available only for reference laboratories
to where thousands of isolates are sent annually from
different hospitals to be serosubtyped.
At present, OMV vaccines are used more widely than
any other serogroup B vaccine in many countries.
According to the most recent studies, the great majority
of OMV vaccines developed, are obtained from epidemic
strains expressing PorA P17 and P119 epitopes on their
outer membranes (Sierra et al. 1991; Boslego et al. 1999;
Rouppe et al. 2000; Vermont et al. 2003; Oster et al.
2005). Therefore, the rapid and accurate incidence determination of these two epitopes among strains causing
invasive disease within a population is essential for health
authorities to take control measures.
The method here proposed proved to be fast, sensitive
and highly specific to determine those VR1 variants. The
total time needed from DNA extraction from the cultured
organism to the result, is about 46 h depending on the
laboratory equipments and staff. Conventional serosuptyping can take 12 days to get the final result. An additional advantage of this method over serosubtyping is the
possibility to be performed directly from samples that
show negative results after conventional diagnostic laboratory methods. This advantage could be crucial to detect
changes in epidemiological patterns of countries where
false-negative results surpass the number of laboratory
confirmed cases like in Brazil. We believe that the
rapid identification of such variants is of the utmost

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429

Rapid detection of N. Meningitidis PorA VRs

importance for rapid determination of circulating types

during outbreaks or even in endemic areas, being a
systematic way of selecting PorA vaccine candidates and
analysing vaccine coverage and failure.
Finally, we believe that the rapid assessment of the
occurrence of P17 and P119 epitopes within porA gene,
could rapidly point to the emergence of antigenic variations among circulating strains.
Acknowledgements
The authors thank the Plataforma Genomica Sequenciamento de DNA PDTIS-FIOCRUZ for the sequencing
reactions. This work was supported by INCQS
FIOCRUZ.
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