Establishment and Modification of Ninety-seven Pneumococcal Serotyping Assays Based on Quantitative Real-time Polymerase Chain Reaction.

Objective: To establish and modify quantitative real-time polymerase chain reaction (qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes. Methods: A database of capsular polysaccharide ( cps) loci sequences was generated, covering 97 pneumococcal serotypes. Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs. A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR, while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay. A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping. Results: A total of 97 pneumococcal serotyping assays based on qPCR were established and modified, which included 64 serotypes previously reported as well as an additional 33 serotypes. Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system. A total of 97 pneumococcal serotypes, which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups, could not be identified as individual serotypes. The sensitivity of qPCR assays based on 27 target sequences was 1-100 copies/µL. The specificity of the qPCR assays was 100%, which were tested by a panel of 90 serotypes of the pneumococcal reference strains. Conclusion: A total of 27 novel qPCR assays were established and modified to analyze 97 pneumococcal serotypes.

Genetic and Phenotypic Variation of Campylobacter jejuni NCTC11168 Caused by flhA Mutation during Laboratory Passage.

Objective: Campylobacter jejuni NCTC11168 is commonly used as a standard strain for flagellar biosynthesis research. In this report, two distinguished phenotypic isolates (CJ1Z, flhA mutant strain, lawn; CJ2S, flhA complemented strain, normal colony) appeared during laboratory passages for NCTC11168. Methods: Phenotypic assessments, including motility plates, transmission electron microscopy, biofilm formation assay, autoagglutination assay, and genome re-sequencing for these two isolates (CJ1Z, flhA mutant strain; CJ2S, flhA complemented strain) were carried out in this study. Results: Transmission electron microscopy revealed that the flagellum was lost in CJ1Z. Phenotypic assessments and genome sequencing of the two isolates were performed in this study. The capacity for biofilm formation, colony auto-agglutination, and isolate motility was reduced in the mutant CJ1Z. Comparative genomic analysis indicated a unique native nucleotide insertion in flhA (nt, 2154) that caused the I719Y and I720Y mutations and early truncation in flhA. Conclusion: FlhA has been found to influence the expression of flagella in C. jejuni. To the best of our knowledge, this is the first study to describe the function of the C-terminal of this protein.

Genetic Characteristics of Lipooligosaccharide and Capsular Polysaccharide of Campylobacter jejuni from Different Sources in China.

Objective: To determine the distribution of two important virulence factors [lipooligosaccharide (LOS) and capsular polysaccharide (CPS)] in Campylobacter jejuni ( C. jejuni) isolated from different sources in China and to develop a rapid screening method for Guillain-Barré syndrome (GBS)-associated strains. Methods: Whole-genome sequencing was carried out for 494 C. jejuni strains. The OrthoMCL software was used to define the LOS/CPS gene clusters. CPS genotyping was performed with serotype-specific sequence alignment using the BLAST software. Real-time Polymerase chain reaction (PCR) was developed with the unique sequences of specific CPS types. Results: Nine novel and 29 previously confirmed LOS classes were identified. LOS classes A, B, and C were the most common (48.2%, 238/494) among the 494 strains. Twenty-six capsular types were identified in 448 strains. HS2, HS4c, HS5/31, HS19, and HS8/17 were the most frequent CPS genotypes (58.7%, 263/448). Strains of 17 CPS genotypes (strain number > 5) had one or two prevalent LOS classes ( P < 0.05). Multiplex real-time PCR for rapid identification of HS2, HS19, and HS41 was developed and validated with strains of known serotypes. Conclusion: Our results describe the genetic characteristics of the important virulence factors in C. jejuni strains in China. The multiplex real-time PCR developed in this study will facilitate enhanced surveillance of GBS-associated strains in China.

Serotype distribution and clinical characteristics associated with streptococcus pneumoniae among Chinese children and adults with invasive pneumococcal disease: a multicenter observational study.

Few studies in China focused on serotypes of Streptococcus pneumoniae in patients with invasive pneumococcal disease (IPD). We aimed at investigating the serotype distribution for IPD-causing S. pneumoniae and vaccine coverage among Chinese children and adults. This was a multicenter, observational study to collect S. pneumoniae isolates from normal sterile sites and IPD-related clinical information among children and adults. Serotyping was performed by a Capsule-Quellung reaction test using type-specific antisera. The study collected a total of 300 eligible isolates (pediatric = 148, adult = 152) were serotyped in a central laboratory. The most prevalent serotypes were 19A (20.9%) and 23 F (20.3%) in the pediatric group; 3 (21.7%) and 19 F (11.8%) in the adult group. PCV10 had low-to-moderate serotype coverage rates for children (60.8%) and adults (34.2%). PCV13 and PPV23 had high coverage rates for children (89.9%, 93.2%) and adults (70.4%, 82.9%), respectively, Investigational PCVs including PCV15 and PCV20 had high estimated coverage rates in children (89.9%, 93.9%). The study identified 269 subjects with IPD reported as the primary diagnosis in the medical records. Sepsis (48/136, 35.3%) and pneumonia (48/133, 36.1%) had the highest occurrence in the pediatric and adult groups, respectively. Study findings showed that non-PCV7 S. pneumoniae 19A and 3 were the most prevalent serotypes in Chinese children and adults, respectively. High-valent vaccines had similar coverage rates and may have a greater potential in preventing IPD.

Comparison of the Pathogenicity of Neisseria meningitidis Isolates of Hyperinvasive Sequence Type 7 Belonging to Serogroups A, B, C, and X.

OBJECTIVE: To compare the pathogenicity of isolates of sequence type 7 (ST-7) Neisseria meningitidis( N. meningitidis) belonging to four different serogroups (A, B, C, and X). METHODS: Four ST-7 N. meningitidis isolates serogrouped as A, B, C, and X and characterized by different capsule structures, were examined for their adhesion and invasion properties, and their ability to induce cytokine release and apoptosis in the host cell (the A549 cell line). RESULTS: Among the four ST-7 N. meningitidis isolates, the serogroup A isolate possessed the strongest adhesion and invasion ability. This isolate also induced the release of the highest levels of the pro-inflammatory mediators interleukin-6, interleukin-1ß, and interferon, and the highest apoptosis rate in the host cells. However, there was no significant difference in interleukin-8 and tumor necrosis factor-α secretion between the four isolates. Based on the findings, the serogroup X N. meningitidis isolate had the weakest pathogenicity, whereas there was almost no difference in the pathogenicity of the isolates from serogroups B and C. CONCLUSIONS: The differences in the capsular structure of the four isolates of ST-7 N. meningitidis affected their pathogenic capacities. The findings also imply that the hyperinvasive ST-7 N. meningitidis lineage may include hypoinvasive isolates.

Lower respiratory tract isolates of non-typeable Haemophilus influenzae in Western Sichuan, China: Antimicrobial susceptibility, mechanisms of ß-lactam resistance and decade changes.

OBJECTIVES: The aims of this study were to analyse the serotypes of epidemic Haemophilus influenzae and changes in mechanisms of ß-lactam resistance over the past decade. RESULTS: Haemophilus influenzae isolates in Western Sichuan from 2013-2014 were non-typeable H. influenzae (NTHi). ß-Lactam MICs for NTHi isolated during 2013-2014 were significantly higher than those from 2003-2004 (P < 0.05). Of 274 NTHi, 141 (51.5%) were ß-lactamase-positive (TEM-1 type). There were 35 amino acid (AA) substitutions in ftsI among NTHi isolated from 2013-2014. However, NTHi isolates from 2003-2004 had only nine AA substitutions. Ordered multiple classification logistic regression analysis showed that different AA substitution patterns in ftsI had different effects on ß-lactam MICs. The main factors affecting the ampicillin MIC were the mutations R517H (OR = 6.999), L389F (OR = 7.128), N526K (OR = 4.660) and D350N (OR = 0.450). The main factor influencing the amoxicillin/clavulanic acid MIC was an N526K mutation (OR = 9.349). The main factors affecting the cefuroxime MIC were the mutations S357N (OR = 37.453) and N526K (OR = 14.816). Compared with 2003-2004, gBLNAR and gBLPAR isolated from 2013-2014 increased significantly from 13.0% (7/54) and 9.3% (5/54) to 38.2% (84/220) and 45.5% (100/220), respectively (P < 0.001). In the 'others' group of ftsI gene mutations, 13 NTHi had the same ftsI gene mutation pattern and 24 AA substitutions. CONCLUSION: These results confirm that ß-lactam-resistant NTHi isolates increased rapidly. AA substitutions in ftsI were more complex and diversified in 2013-2014.

A Cross-sectional Survey Assessing Carriage of Streptococcus pneumoniae in a Healthy Population in Xinjiang Uygur Autonomous Region of China.

The carriage rate and serotype distribution of Streptococcus pneumoniae (S. pneumoniae) in a healthy population in China remains unclear. In this study, we collected the oropharyngeal swabs from 513 individuals in Xinjiang, China. Real-time PCR targeting the lytA gene and 12 serotypes were assessed to identify S. pneumoniae carriage. The total carriage rate of S. pneumoniae was 70.4% (361/513). The most prevalent serotypes were 19B/F, 18B/C, 5, and 6A/B. The highest carriage rate of S. pneumoniae was noted in children aged 6-10 years (88.6%), which merits further attention. The co-colonization rate of two or more S. pneumoniae serotypes was 79.8% (264/331). This study aimed to investigate the baseline pneumococcal carriage rate among healthy individuals in China to improve our understanding of the epidemiology of S. pneumoniae.

[Three quantitative methods to continuously monitor Legionella in spring water].

OBJECTIVE: To compare the detection effect of Legionella pollution in spring water by three methods, namely traditional plating method, fluorescent quantitation PCR method and ethidium monoazide (EMA) fluorescent quantitation PCR method. METHODS: Every month (except May), we collected 11 water samples from the 5 selected hot spring pools in one hot spring resort in Beijing in 2011. A total of 121 water samples were collected, and then were detected by the above three methods qualitatively and quantitatively. RESULTS: In our study, the Legionella pollution rate was separately 74.4% (90/121), 100.0% (121/121) and 100.0% (121/121) by the above three methods. The quantitative value of Legionella in the 121 water samples detected by the three methods were around 0.10-216.00 colony-forming units (CFU)/ml, 1.47-1557.75 gene units (GU)/ml and 0.20-301.69 GU/ml, respectively. The median (25th and 75th percentiles) was 75.30 (32.51-192.10) GU/ml, 36.46 (16.08-91.21) GU/ml and 5.30 (0.00-33.70) CFU/ml, respectively. The difference in the quantitative value of Legionella detected by the three methods showed statistical significance (χ(2) = 187.900, P < 0.01). The quantitative value of Legionella detected by fluorescent quantitation PCR method was the highest, followed by the value Legionella detected by EMA-fluorescent quantitation PCR method and traditional plating method. CONCLUSION: The sensitivity of the PCR methods was higher than traditional plating method, in detecting Legionella pollution in spring water, especially the EMA- fluorescent quantitation PCR method, which was more suitable for detecting Legionella in water.

Molecular characteristics of Neisseria meningitidis isolated during an outbreak in a jail: association with the spread and distribution of ST-4821 complex serogroup C clone in China.

OBJECTIVE: To characterize the meningococcal strains isolated from cases and close contacts with meningococcal disease associated with an outbreak in a jail in May 2010 by investigating the national distribution of hyperinvasive ST-4821 serogroup C clone associated with this outbreak. METHODS: The cases were described based on the clinical symptoms and laboratory results. Pharyngeal swabs were cultured for N. meningitidis from men in the jail. Meningococcal isolates were identified by serogrouping, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), respectively. Four hundred and sixteen serogroup C N. meningitidis strains were collected from 27 provinces between 2003 and 2010 for a nationwide survey and analyzed by PFGE and MLST. RESULTS: Three persons in a jail system were infected with invasive N. meningitidis serogroup C. All isolates tested had matching PFGE patterns and belonged to the multilocus sequence type (ST) 4821 clonal complex. All 47 N. meningitidis strains were identified from the pharyngeal swabs of 166 peoples in the jail, and 26 of them belonged to ST-4821 serogroup C clone, and 90.14% (375/416) serogroup C strains identified in the nationwide survey belonged to the ST-4821 complex. The ST-4821 serogroup C clone was spread nationwide, distributed in 24 provinces, especially in eastern provinces between 2003 and 2010. CONCLUSION: Endemic transmission and carriage rate of ST-4821 serogroup C clone are high in this jail system. The ST-4821 serogroup C clone is spreading in China and nationwide distributed despite the existence of some effective vaccines.

[Comparison on the levels of human serum antibody against Neisseria meningitidis serogroup C measured using serum bactericidal assay and ELISA].

OBJECTIVE: To analyze the levels of human serum antibody against Neisseria meningitidis serogroup C measured by serum bactericidal assay (SBA) and ELISA. METHODS: SBA and a modified ELISA were applied to measure the serum bactericidal titer and the specific concentration of immunoglobulin G (IgG) against meningococcal serogroup C in sera samples. Seventy-five sera were from healthy adults without undertaking vaccination while another 429 and 388 pre- and post-vaccinated sera were from 143 infants and 194 young children immunized with conjugate vaccine or polysaccharide vaccine, respectively. Correlation between serum bactericidal titer and the concentration of specific IgG against meningococcal serogroup C was analyzed. RESULTS: The concentration of meningococcal serogroup C specific IgG in healthy adults showed a strong correlation (r=0.814 33, P<0.001) with serum bactericidal titer through linear regression analysis. Weak correlation was observed between SBA titers and IgG concentration in pre vaccinated sera of infants and children (conjugate/polysaccharide vaccine) (infants: r=0.140 64, P>0.100/r=0.2899, P<0.05; children: r=0.540 40, P<0.05/r=0.194 36, P<0.05). After immunization with 2-dose conjugate vaccine in infants and 1-dose in children, a strong correlation between the two panels of results was observed (r=0.809 38, P<0.001 and r=0.837 23, P<0.001 respectively). However after immunization with polysaccharide vaccine, the correlation between serum bactericidal titer and concentration of specific IgG was weak (r<0.50000). CONCLUSION: Among healthy adults and post vaccinated infants or young children immunized with conjugate vaccine, the concentration of specific IgG was comparable to the serum bactericidal titer against meningococcal serogroup C. However, it was not unfavorable to use ELISA as the principal means of measuring serum antibody responses to polysaccharide vaccine for infants under 1 year old.

[Contamination status and molecular biological characteristics of Legionella in cooling water samples from different places in Wuxi city].

OBJECTIVE: To investigate the contamination state of Legionella in cooling water samples from different places in Wuxi city and reveal the molecular biological characteristics of Legionella strains. METHODS: 112 parallel water samples (500 ml each) were collected from 56 sites in Wuxi city during year 2009 - 2010. The samples were used for Legionella test and quantitative culture. The isolated Legionella strains were used for serotyping, pulsed-field gel electrophoresis (PFGE), sequence-based typing (SBT), and intracellular growth were tested. RESULTS: The positive proportion of Legionella was 39. 3% (22/56) among all sampling sites. A total of 29 Legionella strains were isolated, and the serotypes include LP1, LP3, LP5 and LP6. LP1 serotype was the major one with a proportion of 65.5% (19/29). 29 Legionella strains got 17 PFGE types. There were 10 SBT types among 10 Legionella strains with different PFGE types. Comparing to LP1 strain (ATCC 33152), WX2011062 (LP6) and WX2011067 (LP5) had strong intracellular growth ability in mouse peritoneal macrophages J774 cell line (the amount of intracellular bacteria on day 0 after infection were (5.5 +/- 1.32) x 10(5), (3.9 +/- 0.60) x 10(5), (7.8 +/- 0.76) x 10(5) CFU/ml, respectively; the amount of intracellular bacteria on day 3 after infection were (58.3 +/- 1.61) x 10(5), (2700.0 +/- 655.74) x 10(5), (3066.7 +/- 208.17) x 10(5) CFU/ml, respectively). CONCLUSION: The Legionella contamination existed in cooling water samples from different places in Wuxi city. Legionella strains isolated showed high genetic variation. Some Legionella strains had vigorous intracellular growth ability.

[Molecular typing of Legionella pneumophila isolates from China with pulsed field gel electrophoresis].

OBJECTIVE: To analyze the molecular types of Legionella (L.) pneumophila strains isolated in China, and to develop the PulseNet-China Database of L. pneumophila. METHODS: Pulsed field gel electrophoresis (PFGE) was used to analyze 262 L. pneumophila strains collected from 11 provinces between 2004 and 2009 in China. Different kinds of genomic DNA in different L. pneumophila strains were isolated and separated after digesting with Asc I. BioNumerics software was used to analysis the PFGE fingerprints. RESULTS: L. pneumophila strains isolated in China were quite different regarding their PFGE patterns. There were 108 PFGE types among the 262 strains tested in this study. The similarity value of these strains was in the range of 16% - 100% and the same types were discovered in different provinces and years. CONCLUSION: L. pneumophila strains isolated in China were with high genetic variations. There might be different clones existed in China. The development of PulseNet China Database was thus of great significance in monitoring the L. pneumophila strains in the future.

[Sequence-based typing of 82 strains of serotype I Legionella pneumophila isolated from 9 provinces in China].

OBJECTIVE: To analyze the characteristics of Sequence-based Typing (SBT) of the Serotype 1 Legionella pneumophila (Lp1) isolated from environmental water in China, and then create a preliminary database. METHODS: A total of 82 strains of Lp1 isolated from environmental water in 9 provinces of China between 2005 and 2008 were genotyped by SBT method and Pulsed-field Gel Electrophoresis (PFGE) method. The results of the two different typing methods were then compared by cluster analysis, adopting BioNumerics version 5.1 software. RESULTS: By SBT method, the 82 strains of Lp1 were divided into 22 ST types, of which 17 new types and one new allele was discovered. The dominant type was ST-1 type, found in 8 provinces, accounting for 46.3% (38/82). ST-1, ST-150, ST-154, ST-159, ST-160 and ST-630 types were found in more than 2 isolated-sites; while more than 2 different ST types were found in 5 isolated-sites, as site B4, B5, B6, S3 and S8. In cluster analysis, 15 ST types were grouped into three complexes (ST-1 complex, ST-154 complex and ST-149 complex); and the other 7 ST types were not assigned complex. By PFGE method, 46 banding patterns were observed. As a result of the combination of the two methods, the 82 isolates strains could be divided into 54 molecular types, which showed a reliable accordance in the cluster analysis between the two methods. CONCLUSION: The SBT of the Lp1 in environmental water in China was unique. From the study, a preliminary SBT database was set up.

[Genotypic variability and persistence of Legionella pulsed-field gel electrophoresis patterns in 16 cooling towers in Shanghai, China].

OBJECTIVE: To investigate the genotypic characteristics and persistence of Legionella pulsed-field gel electrophoresis (PFGE) patterns in 16 air-conditioner cooling towers in six different public sites of Shanghai. METHODS: From May to October, continuous sampling was operated once per month in 2007. Legionella strains isolated from the 16 cooling towers were confirmed by serological and latex agglutination. PFGE was applied for the fingerprinting of the isolates, while the cluster results of PFGE were analyzed by BioNumerics software. RESULTS: 131 strains of Legionella were isolated, including L. pneumophila, L. bozemanae, L. micdadei and L. anisa. 52 distinguishable PFGE patterns were differentiated among the 16 cooling towers, with 37 patterns were owned by just one cooling tower, which was not shared with other cooling towers, while 15 patterns were shared by more than 2 cooling towers. All the cooling towers had ≥ 2 PFGE patterns, while in 13 cooling towers the same PFGE patterns were recovered during the six months. From June to October of 2007, 18 strains of Legionella belonging to the PFGE pattern of LPAs.SH0078 were isolated continuously from 6 cooling towers. CONCLUSION: This study demonstrated great genotypic diversity and complexity of Legionella in cooling towers. Persistence of the PFGE patterns was observed in 81.25% of the cooling towers. The PFGE pattern of LPAs. SH0078 was distributed widely, suggesting it might be the dominate strain in Shanghai.

[Molecular typing of Klebsiella pneumonia by pulse-field gel electrophoresis in combination with multilocus sequence typing].

OBJECTIVE: To type Klebsiella pneumonia through methods including pulse-field gel electrophoresis (PFGE) in combination with multilocus sequence typing. METHODS: Four selected different EPs, referring to the Standard Operating Procedure of PulseNet China, were used. The single colony of Klebsiella pneumonia was quantified after enriched culture. Embedding organisms in agarose and genome DNA were lysed with Proteinase K and then digested by restriction endonuclease XbaI, to produce agarose gel. Fingerprint was obtained through PFGE and bands were marked with their molecular weights and then analyzed by BioNumerics software. Using MLST to analyze the strains that were highly similar, by PFGE typing RESULTS: By comparing the four results from each EPs, fk3 (switch time from 6s to 36s, total run time is 18.5 hours) seemed to be better than the others. 59 strains of Klebsiella pneumonia were divided into 47 PFGE types and 19 PFGE clusters. The highly similar strains could be typed into ST-340, ST-342, ST-343, ST-344, ST-345 by MLST. Among them, ST-342, ST-343, ST-344, ST-345 types were all new MLST types that were reported in China. CONCLUSION: Highly similar Klebsiella pneumonias typed by PFGE could also be typed by MLST.

Proteome analysis of Neisseria meningitidis serogroup strains C associated with outbreaks in China.

OBJECTIVE: During 2003-2005, an outbreak of meningitis due to Neisseria meningitidis serogroup C occurred in China. With the aim to find strain clues result in the final epidemics, the ancestral strain 053442, a clinical isolate, and a carrier strain 053426 with different gene type were analyzed. METHODS: Clinical strain 053442 and carrier strain 053426 were cultured on GC agar plates under the same condition. Two-dimensional electrophoresis was performed using the pH 3-10 nonlinear IPG strips of 24 cm length, and all the protein spots were identified by matrix-assisted laser desorption/ionization time of flight spectrometry. RESULTS: 502 and 380 protein spots were identified in 053426 and 053442 respectively, relating to 266 and 202 different genes covering a wide range of cellular functions. The express volume and number of proteins involved in energy metabolism, protein synthesis and amino acid biosynthesis in 053426 were higher than in 053442. Virulence factor Opa, Opc and a series of proteins involved in pilus assembly and retraction were identified in 053442, which appear to be of primary importance in colonization and invasion of human cells. Compared to 053442, virulence protein species were less in 053426, with lower express volumes too. No Opa and Opc were detected in 053426. CONCLUSIONS: The different protein expression profiles of the clinical strain 053442 and carrier strain 053426 in the present study provide some clues of the different pathogenicity of the two strains, which may account for result in the final epidemics.

[Establishment and application of PCR method in serotyping of Streptococcus pneumonia.].

OBJECTIVE: To establish a method based on PCR for serotyping of Streptococcus pneumonia isolates. PCR serotyping method was applied for investigating the serotypes of S. pneumonia strains. METHODS: 12 pairs of primers targeting different serotypes of S. pneumonia were designed and synthesized. After optimizing the PCR amplification reaction, sensitivity and specificity of each pair was performed. We applied the PCR methods for testing the serotypes of the isolated S. pneumonia strains. RESULTS: Each pair of primers showed satisfied PCR sensitivity and specificity. Of all 119 S. pneumonia strains tested by PCR serotyping method, 113 isolates were identified (3, 5, 6A/B, 9A/V, 14, 18, 19A, 19F, 23F) with 6 isolates were unable to be serotyped. CONCLUSION: We developed a simple, reliable and economic method for S. pneumonia serotyping which could be used for testing the serotypes of S. pneumonia that had been prevailed among general population.

[Study on the bactericidal antibody against Neisseria meningitidis serogroup C strains after immunization with a divalent polysaccharide (A plus C) vaccine].

OBJECTIVE: To optimize the serum bactericidal assay (SBA), detect and analyze the bactericidal antibody level against Neisseria meningitidis serogroup C strains after divalent polysaccharide (A plus C) vaccine immunization. METHODS: Two Neisseria meningitidis serogroup C strains, vaccine candidate strain (C11) and epidemic strain (053442), were selected as targets. The national Neisseria meningitidis standardized serum was used as reference serum. Pel-Freez infant rabbit complements was available. The optimized SBA method was used to detect bactericidal antibody against strain C11 and 053442 for 122 pairs of sera before and after immunization with a divalent polysaccharide (A and C) vaccine. RESULTS: The strain C11 and 053442 both could be used as targeted strain for SBA. The optimized concentration of targeted strain was achieved when a whole-cell suspension of 0.35 A at 600 nm was diluted 4 x 10(4) times. Before immunization, SBA geometric mean titers (GMT) of 122 sera against strain C11 and 053442 were 1:1.75 and 1:2.63 respectively, and the protective rates were 9.8% and 17.2% respectively. After immunization, the GMTs and the protective rates of 122 sera both rose significantly (P<0.01), the GMTs against strain C11 and 053442 were 1:483.73 and 1:412.57 respectively. The protective rates against strain C11 and 053442 were 100% and 95.9% respectively. CONCLUSION: Immunization with a divalent polysaccharide (A and C) vaccine could elevate remarkably the population SBA titer against Neisseria meningitidis serogroup C strains of different subtypes, but the surveillance of vaccine effect against different targeted strains remains necessary.