Neisseria meningitidis Strain of Unknown Serogroup, China

To the Editor: Neisseria meningitidis is a major public health hazard in many parts of the world. This organism is classified into 13 serogroups, and most meningococcal disease is caused by strains that express 1 of the 5 types of capsular polysaccharides (A, B, C, Y, and W135). In the natural reservoir of the human nasopharynx, strains of N. meningitidis that do not fit into 1 of the 13 serogroups and are presumably unencapsulated are common. By contrast, rare meningococcal diseases are caused by these nonserogroupable strains. In this article, we describe a case of N. meningitidis infection caused by a nonserogroupable strain in the People’s Republic of China and the genotype characteristics of this strain. 
 
The patient was a 6-month-old boy who was admitted to a local hospital in Beijing in May 2009. The infection started suddenly with high fever (39°C). N. meningitidis infection was confirmed on the basis of the clinical signs and results of laboratory examination. Nausea, vomiting, and neck stiffness developed, and the patient lost consciousness. Physical examination showed a positive Kernig sign and negative Brudzinski sign. The patient’s cerebrospinal fluid sample was injected into chocolate agar, in which microbial growth was observed after 24 hours. The API NH system (bioMerieux, Marcy-Etoile, France) showed that the isolate was N. meningitidis. However, this strain could not be placed in a serogroup, even after specific antiserum (Remel, Lenexa, KS, USA) was used. No other disease with complement deficiency was detected in the patient. The patient’s infection was treated with antimicrobial drugs, and he recovered completely. 
 
We investigated this nonserogroupable N. meningitidis strain by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and subtyping of the variable regions of the genes (porA, porB, and fetA) encoding the outer membrane proteins. MLST indicated that the fumC was a new allele with a new number 482. The allele numbers for abcZ, adk, fumC, gdh, pdhC, and pgm were 222, 3, 58, 386, 18, and 77, respectively. This strain was assigned a new sequence type number, ST7962. 
 
Among the 44 complexes designed in the MLST database, ST7962 was most similar to the ST4821 complex with 3 identical loci. The PFGE pattern of this strain was compared with the PFGE patterns in the reference database of N. meningitidis from China by using BioNumerics version 5.10 software (Applied Maths, Kortrijk, Belgium). At the time of comparison, the database contained 618 isolates of N. meningitidis and 243 PFGE patterns. This strain had a single pattern that was clustered together with the ST4821 complex strains in the cluster tree based on the PFGE patterns. The PorA genotype of the strain was determined to be P1.7–2, 14, which was a genotype associated with the ST4821 complex serogroup C strains that caused outbreaks in China in 2003 (1). The porB and fetA alleles of this strain were 3–18 and F4–21, respectively. 
 
The genetic basis for the reason that this strain was nonserogroupable was studied by PCR and sequencing. PCR showed that this strain had intact capsule genetic islands of ctrA-D, synA-C, lipA, and lipB, and contained synD, encoding the serogroup B polysialyltransferase. The capsular gene clusters were sequenced entirely, and a missense mutation within synD was identified (Figure). The mechanism underlying the capsule phase variation of N. meningitidis serogroup B involves a variation caused by slipped-strand mispairing in the polyC tract at the 5′ end of synD (2–4). A tract of 7 C residues encodes capsular expression, and an insertion or deletion of 1 C results in a missense mutation within synD, thereby leading to nonexpression of the capsule. Nucleotide sequencing of synD of our isolate revealed an insertion of 1 C within the polyC tract. Thus, slipped-strand mispairing within synD was predicted to be the mechanism underlying the nonserogroupability of this strain. 
 
 
 
Figure 
 
Genetic basis for the Neisseria meningitidis strain that cannot be placed in a known serogroup. A predicted slipped-strand mispairing occurred within synD, which encodes the serogroup B sialyltransferase. In wild-type N. meningitidis serogroup B (MC58), ... 
 
 
 
Few reports have described invasive meningococcal disease caused by nonserogroupable N. meningitidis strains; the lack of such reports suggests that complement deficiency might be a predisposing factor, and all the reported isolates were determined to be capsule null locus (cnl) strains, which lacked the genetic islands encoding the entire capsule (5–7). However, the patient described here was not found to have a complement deficiency, and the disease-associated nonserogroupable N. meningitidis strain in this study contained the genetic locus of an intact capsule. 
 
In China, meningococcal polysaccharide vaccines A and C have been used for routine immunization. In many countries in Africa, repeated vaccination against N. meningitidis serogroups A and C have likely led to a selective increase in the incidence of meningococci of other serogroups, thereby resulting in a changed profile of meningococcal disease (8). In recent years, invasive disease caused by N. meningitidis serogroup W135 and serogroup X strains has emerged in China (9,10). Therefore, meningococcal disease caused by serogroups other than A and C as well as nonserogroupable N. meningitidis strains appears to be an emerging problem and should be investigated epidemiologically.


Neisseria meningitidis Strain of Unknown Serogroup, China
To the Editor: Neisseria meningitidis is a major public health hazard in many parts of the world. This organism is classifi ed into 13 serogroups, and most meningococcal disease is caused by strains that express 1 of the 5 types of capsular polysaccharides (A, B, C, Y, and W135). In the natural reservoir of the human nasopharynx, strains of N. meningitidis that do not fi t into 1 of the 13 serogroups and are presumably unencapsulated are common. By contrast, rare meningococcal diseases are caused by these nonserogroupable strains. In this article, we describe a case of N. meningitidis infection caused by a nonserogroupable strain in the People's Republic of China and the genotype characteristics of this strain.
The patient was a 6-month-old boy who was admitted to a local hospital in Beijing in May 2009. The infection started suddenly with high fever (39°C). N. meningitidis infection was confi rmed on the basis of the clinical signs and results of laboratory examination. Nausea, vomiting, and neck stiffness developed, and the patient lost consciousness. Physical examination showed a positive Kernig sign and negative Brudzinski sign. The patient's cerebrospinal fl uid sample was injected into chocolate agar, in which microbial growth was observed after 24 hours. The API NH system (bioMérieux, Marcy-Etoile, France) showed that the isolate was N. meningitidis. However, this strain could not be placed in a serogroup, even after specifi c antiserum (Remel, Lenexa, KS, USA) was used. No other disease with complement defi ciency was detected in the patient. The patient's infection was treated with antimicrobial drugs, and he recovered completely.
We investigated this nonserogroupable N. meningitidis strain by multilocus sequence typing (MLST), pulsed-fi eld gel electrophoresis (PFGE), and subtyping of the variable regions of the genes (porA, porB, and fetA) encoding the outer membrane proteins. MLST indicated that the fumC was a new allele with a new number 482. The allele numbers for abcZ, adk, fumC, gdh, pdhC, and pgm were 222, 3, 58, 386, 18, and 77, respectively. This strain was assigned a new sequence type number, ST7962.
Among the 44 complexes designed in the MLST database, ST7962 was most similar to the ST4821 complex with 3 identical loci. The PFGE pattern of this strain was compared with the PFGE patterns in the reference database of N. meningitidis from China by using BioNumerics version 5.10 software (Applied Maths, Kortrijk, Belgium). At the time of comparison, the database contained 618 isolates of N. meningitidis and 243 PFGE patterns. This strain had a single pattern that was clustered together with the ST4821 complex strains in the cluster tree based on the PFGE patterns. The PorA genotype of the strain was determined to be P1.7-2, 14, which was a genotype associated with the ST4821 complex serogroup C strains that caused outbreaks in China in 2003 (1). The porB and fetA alleles of this strain were 3-18 and F4-21, respectively.
The genetic basis for the reason that this strain was nonserogroupable was studied by PCR and sequencing. PCR showed that this strain had intact capsule genetic islands of ctrA-D, synA-C, lipA, and lipB, and contained synD, encoding the serogroup B polysialyltransferase. The capsular gene clusters were sequenced entirely, and a missense mutation within synD was identifi ed (Figure). The mechanism underlying the capsule phase variation of N. meningitidis serogroup B involves a variation caused by slipped-strand mispairing in the polyC tract at the 5′ end of synD (2)(3)(4). A tract of 7 C residues encodes capsular expression, and an insertion or deletion of 1 C results in a missense mutation within synD, thereby leading to nonexpression of the capsule. Nucleotide sequencing of synD of our isolate revealed an insertion of 1 C within the polyC tract. Thus, slipped-strand mispairing within synD was predicted to be the mechanism underlying the nonserogroupability of this strain.
Few reports have described invasive meningococcal disease caused by nons erogroupable N. meningitidis strains; the lack of such reports suggests that complement defi ciency might be a predisposing factor, and all the reported isolates were determined to be capsule null locus (cnl) strains, which lacked the genetic islands encoding the entire capsule (5)(6)(7). However, the patient described here was not found to have a complement defi ciency, and the disease-associated nonserogroupable N. meningitidis strain in this study contained the genetic locus of an intact capsule.
In China, meningococcal polysaccharide vaccines A and C have been used for routine immunization. In many countries in Africa, repeated vaccination against N. meningitidis serogroups A and C have likely led to a selective increase in the incidence of meningococci of other serogroups, thereby resulting in a changed profi le of meningococcal disease (8). In recent years, invasive disease caused by N. meningitidis serogroup W135 and serogroup X strains has emerged in China (9,10). Therefore, meningococcal disease caused by serogroups other than A and C as well as nonserogroupable N. meningitidis strains appears to be an emerging problem and should be investigated epidemiologically. Genetic basis for the Neisseria meningitidis strain that cannot be placed in a known serogroup. A predicted slipped-strand mispairing occurred within synD, which encodes the serogroup B sialyltransferase. In wild-type N. meningitidis serogroup B (MC58), the synD polyC tract contains 7 C residues, and capsule is expressed. When an insertion (as in isolate 100924) of 1 C residue occurs, a result of local denaturation and mispairing followed by replication or repair, a premature stop codon is generated, and the capsule is not expressed.