A first meningococcal meningitis case caused by serogroup X Neisseria meningitidis strains in China

Neisseria meningitidis is the leading cause of bacterial meningitis and classified into 13 serogroups based on the immunological reactivity of the capsular polysaccharide.1 Serogroups A, B, C, W135 and Y are the most common causes of meningitis.2 Among them, serogroup A and C are the major causes of epidemics in Africa and Asia.2 Most of the epidemic outbreaks of meningococcal meningitis are caused by serogroup A Neisseria meningitidis strain from the 1950s to the 1980s in China.3 During the years 2003 and 2005, a new sequence type (ST-4821) of serogroup C was identified in the Anhui and 11 other provinces of China.4 To well understand the molecular epidemiology of meningococcal meningitis in China, National Institute for Communicable Disease Control and Prevention of China started up a surveillance program for isolating pathogens from the clinical specimens of meningitis patients in 2005. The results of this surveillance program in Beijing, China have been described.5,6 In this study, we reported a sporadic meningococcal meningitis case that was caused by serogroup X Neisseria meningitidis strains. CASE REPORT Symptoms and sample collection A sporadic meningococcal meningitis case was reported to the Beijing Center for Disease Control and Prevention (CDC) on January 11, 2007. The patient was a 14-year-old male student. The onset of this case started quickly with high fever (39°C) and headache on January 11, 2007. Other clinical symptoms included nausea, vomiting, stiff neck and confusion. There was little petechiate rash emerged on the patient’s four limbs. The Kernig’s sign was positive and Brudzinski’s sign was negative. The numbers of white cell in the blood and cerebrospinal fluid (CSF) were 3.6×1010/L and 1.7×109/L, respectively. This case was diagnosed as meningococcal meningitis based on the clinical symptoms. The clinical specimens, including the CSF and blood samples, were collected on January 12, 2007 in Beijing Chaoyang Hospital and sent to the confirmation laboratory of Beijing CDC. PCR amplification DNA from CSF and blood specimens was extracted according to the manual of the QIAamp DNA blood mini kit (QIAGEN, Germany) and detected by multiplex- PCR.7 Two other sets of primers were also designed for the PCR amplification of IS 1106 (P1: 5′-ATTATTCA- GACCGCCGGCAG-3′ and P2: 5′-CCGATAATCAGGC- ATCCG-3′) and ctrA (P3: 5′-GCTGCGGTAGGTGG- TTCAA-3′ and P4: 5′-TTGTCGCGGATTTGCAACTA-3′) fragments of Neisseria species. The products of PCR were analyzed by electrophoresis on a standard 1.5% agarose gel. PCR results are shown in Figure. A 596-bp size IS 1106 fragment (Lane 3) and a 111 bp size ctrA fragment (Lane 4) were amplified from CSF, respectively. The PCR amplifications of IS 1106 and ctrA using the DNA from blood were negative. Both of the multiplex-PCR amplifications with DNA from the CSF and blood were negative. It was concluded that this case was caused by the Neisseria species based on the results of PCR detections. It was unknown which was the causative serogroup of this case.Figure.: PCR amplification of the specific DNA fragments of 5 different serogroups Neisseria meningitidis from the blood and CSF specimens. Lane M: 100-bp DNA ladder. Lane 1: negative control. Lanes 2 and 5: multiplex-PCR amplification of orf-2 (serogroup A) and siaD (serogroups B, C, W135 and Y) fragments. Lanes 3 and 6: PCR amplification of IS 1106 fragments with P1 and P2. Lanes 4 and 7: PCR amplification of ctrA fragments with P3 and P4.Pathogen isolation and identification CSF was also used for pathogen isolation. It was centrifuged for 20 minutes at 2000 r/min. Sediment was vortexed and streaked on 5% sheep blood agar plate. The agar plates were incubated in a 5% CO2 incubator at 37°C overnight. The oropharyngeal swab specimens from the close contacts were obtained and streaked on the 5% sheep blood agar plate for pathogen isolation. Isolates were identified via oxidase test, agglutination with serogrouping antisera, and carbohydrate utilization assays according to the WHO manual.2 The cultures of oropharyngeal swab specimens from the patient’s parents were negative. The culture of CSF was positive and the isolate was identified according to the methods of WHO manual. The oxidase test gave a positive reaction. The agglutination only occurred with the serogroup X antiserum, but not with other serogroup antisera. Carbohydrate utilization assay was further used to validate the identification of this isolate as Neisseria meningitides strains. This isolate oxidized glucose and maltose, but not lactose and sucrose. So this isolate belonged the serogroup X Neisseria meningitidis strains. This result was consistent with that of multiplex-PCR amplification. The reason for the negative multiplex-PCR amplification with the DNA from CSF was that PCR-based assays were routinely used for the detection of serogroups A, B, C, W135, and Y, not for serogroup X and other serogroups. So this case was caused by serogroup X Neisseria meningitidis strain. Molecular epidemiology study The confirmed isolate was used for the molecular epidemiology study with the serotyping, serosubtyping and multilocus sequence typing (MLST) methods. The serotyping and serosubtyping were performed according to the methods described by Sacchi et al.8,9 The full-length DNA fragments of porA and porB genes were amplified and sequenced. The predicted amino acid sequences of PorA and PorB proteins were compared with sequences in the porA and porB database (Neisseria porA and porB typing website http://neisseria.org/nm/typing/) to determine the serosubtype and serotype. MLST was performed as described by Morelli et al10 and Maiden et al.11 The obtained sequences were compared with sequences in the MLST database to determine the allele number (Neisseria multilocus sequence typing website http://pubmlst.org/neisseria/). The sequence type of this strain was determined from the profiles of the seven alleles in the MLST database. The sequence query of porA sequence in the porA database indicated that the numbers of VR1 and VR2 were 18-1 and 3, respectively. The serosubtype of this isolate belonged P1.18-1, 3. The alignment of porB sequence in the porB database indicated that this porB belonged the class 2 outer membrane protein and it was a new porB2 allele sequence. This sequence was submitted to porB database and it was assigned a new allele number 129. The Amino acid sequences for VR1 to 4 of porB protein were “EVSRVKNQGVKTKTATQI”, “KYADL- NTDAERVAVNTANAHSVKDY”, “AAKNNTGTVGE- QGKKHEQTQ”, and “KVNGVKAADNKYQYDQ”. Seven gene fragments, including abcZ, adk, aroE, fumC, gdh, pdhC, and pgm were amplified and sequenced with the ABI 3100 DNA Sequencer (Applied Biosystems, USA). The obtained sequences were compared with sequences in the MLST database to determine the allele number. The result indicated that the aroE was a new allele that was submitted to the database and assigned a new number 457. The other allele numbers for abcZ, adk, fumC, gdh, pdhC, and pgm were 11, 4, 17, 5, 24 and 2, respectively. This sequence type of this isolate was assigned a new number ST-5944. Antibiotic susceptibility The antibiotic susceptibility test was performed by the broth microdilution method according to the protocol of the Clinical and Laboratory Standards Institutes.12 The MICs for ampicillin, azithromycin, cefotaxime, ceftriaxone, chloromycetin, ciprofloxacin, levofloxacin, meropenem, penicillin G, rifampicin, tetracycline and trimethoprim/sulfamethoxazole were 0.25 g/ml, 0.25 μg/ml, 0.03 μg/ml, 0.015 μg/ml, 1 μg/ml, 0.03 μg/ml, 0.03 μg/ml, 0.03 μg/ml, 0.03 μg/ml, 0.12 μg/ml, 0.06 μg/ml and 0.25/4.8 μg/ml, respectively. So this isolate were susceptible to most of the antibiotic, except ampicillin and trimethoprim/sulfamethoxazole. This isolate was medium susceptibility to ampicillin and trimethoprim/sulfamethoxazole. The patient was treated by penicillin G with a favorable outcome. DISCUSSION This study showed a first reported meningococcal meningitis case caused by serogroup X Neisseria meningitides strains in China. Serogroup X Neisseria meningitidis strains were described in the 1960s.13,14 North America, Europe, Australia and Africa also had the serogroup X meningococcal disease cases and outbreaks reports.15-19 Although serogroups A, B, and C were responsible for 90% of meningococcal disease worldwide, recent outbreaks of serogroup X meningococci indicated that serogroup X strain had the capability to cause epidemics. In China, polysaccharide vaccine for serogroup A plus C was usually used for prevention and control of the meningococcal meningitis. Gagneux et al19 reported that repeated vaccination against serogroups A and C in many African countries has the potential to select meningococci of other serogroups (e.g., serogroup X and W 135) and might result in a changed profile of meningococcal disease. The introduction of a new sequence type of serogroup X strain into a susceptible population also might contribute to an epidemic. So it is necessary to develop the comprehensive conjugate vaccines that include capsular polysaccharides from formerly rare causes of disease such as serogroup X. In addition, we will especially monitor the evolution of this clone of serogroup X strain that is responsible for this clinical case.