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Ma-Chao Li,1,* Xiao-Yue Wang,1,* Tong-Yang Xiao,2,* Shi-Qiang Lin,3 Hai-Can Liu,1 Cheng Qian,4 Da Xu,1 Gui-Lian Li,1 Xiu-Qin Zhao,1 Zhi-Guang Liu,1 Li-Li Zhao,1 Kang-Lin Wan1 1State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People’s Republic of China; 2Department of Clinical Laboratory, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, Guangdong, People’s Republic of China; 3Department of Bioinformatics, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian Province, People’s Republic of China; 4Beijing Center for Disease Control and Prevention, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Li-Li Zhao; Kang-Lin Wan, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, P.O. Box 5, Changping, Beijing, 102206, People’s Republic of China, Tel/Fax +86 10 58900779, Email zhaolili@icdc.cn; wankanglin@icdc.cnObjective: To assess the relationship between the variant rpoB mutations and the degree of rifampin (RIF)/rifabutin (RFB) resistance in Mycobacterium tuberculosis (M. tuberculosis).Methods: We analyzed the whole rpoB gene in 177 M. tuberculosis clinical isolates and quantified their minimum inhibitory concentrations (MICs) using microplate-based assays.Results: The results revealed that of the 177 isolates, 116 were resistant to both RIF and RFB. There were 38 mutated patterns within the sequenced whole rpoB gene of the 120 isolates. Statistical analysis indicated that mutations, S450L, H445D, H445Y, and H445R, were associated with RIF and RFB resistance. Of these mutations, S450L, H445D, and H445Y were associated with high-level RIF and RFB MIC. H445R was associated with high-level RIF MIC, but not high-level RFB MIC. D435V and L452P were associated with only RIF, but not RFB resistance. Q432K and Q432L were associated with high-level RFB MIC. Several single mutations without statistical association with rifamycin resistance, such as V170F, occurred exclusively in low-level RIF but high-level RFB resistant isolates. Additionally, although cross-resistance to RIF and RFB is common, 21 RIF-resistant/RFB-susceptible isolates were identified.Conclusion: This study highlighted the complexity of rifamycin resistance. Identification of the rpoB polymorphism will be helpful to diagnose the RIF-resistant tuberculosis that has the potential to benefit from a treatment regimen including RFB.Keywords: Mycobacterium tuberculosis, rifampin resistance, rifabutin resistance, mutation

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.

To evaluate the immunogenicity ofProtein extracts fromImmunization withThis is the advanced study to investigate the immunogenicity of

Li-Li Zhao,*,1 Ming-Xiang Huang,*,2 Tong-Yang Xiao,1 Hai-Can Liu,1 Ma-Chao Li,1 Xiu-Qin Zhao,1 Zhi-Guang Liu,1 Yi Jiang,1 Kang-Lin Wan11State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, People’s Republic of China; 2Clinical Laboratory, Fuzhou Pulmonary Hospital, Fuzhou, 350008, People’s Republic of China*These authors contributed equally to this workObjectives: To explore the prevalence, risk and genetic characteristics of drug-resistant tuberculosis (TB) from a tertiary care TB hospital in China.Patients and methods: We carried out a retrospective study including isolates from 189 patients with pulmonary TB at Fuzhou Pulmonary Hospital. All isolates from these patients were subjected to drug susceptibility testing and genotyping. For drug-resistant isolates, DNA sequencing was used to investigate mutations in 12 loci, including katG, inhA, oxyR–ahpC, rpoB, rpsL, rrs1 (nucleotides 388–1084 of rrs), embB, tlyA, eis, rrs2 (nucleotides 1158–1674 of rrs), gyrA and gyrB.Results: Among 189 isolates, 28.6% were resistant to at least one of the seven anti-TB drugs, including isoniazid (INH), rifampin (RIF), streptomycin (STR), ethambutol (EMB), capreomycin (CAP), kanzmycin (KAN) and ofloxacin (OFX). The proportion of multidrug-resistant TB and extensively drug-resistant TB isolates was 9.5% and 1.1%, respectively. Patients in rural areas as well as previously treated patients showed a significantly increased risk of developing drug resistance. In addition, among these isolates, 111 (58.7%) were Beijing genotype strains, 84 (75.7%) of which belonged to modern Beijing sublineage. There was no association between genotype and drug resistance. The most common mutations were katG315, rpoB531 rpsL43, embB306, rrs1401 and gyrA94. Conclusion: These findings provided additional information of drug-resistant TB in China. Previously treated patients and patients in rural areas should receive greater attention owing to their higher risk of developing drug resistance.Keywords: drug-resistant tuberculosis, genotype, risk, mutation

This study aimed to characterize the diagnostic and vaccine potential of a novel Mycobacterium tuberculosis antigen Rv0674.To evaluate the diagnostic potential and antigenicity of Rv0674, IgG was evaluated using ELISA and interferon (IFN)-γ was done by using ELISpot assay among TB patients and healthy donors. For immunogenicity evaluation, BALB/c mice were immunized with Rv0674. Cytokine production was determined by cytokine release assay using an ELISA kit, and the antibodies were tested using ELISA.The results of serum Elisa tests showed that Rv0674 specific immunoglobulin G (IgG) response was higher in TB patients than negative controls. And Rv0674 had good performance in serological test with sensitivity and specificity of 77.1% and 81.1%, respectively. While it shows poor sensitivity and specificity of 26.23% and 79.69% for IFN-γ tests. In BALB/c mice, Rv0674 adjuvant by DDA/Poly I:C could also induce a high level of IFN-γ, interleukin-2 and interleukin-6 as well as a high IgG titer in both high- and low-dose groups indicating that Rv0674 is essential in humoral and cellular immunity. Moreover, the cytokine profile and IgG isotype characterized Rv0674 as a Th1/Th2-mixed-type protective immunity with the predominance of Th1 cytokines.Rv0674 may be a good potential candidate for the development of TB serological diagnosis and a new TB vaccine.

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.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.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, P0.01). 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.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.

Microstructures and phase equilibria in cast and annealed Nb–Mo–Si alloys near the Nb–Mo binary were studied using X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), and electron beam microprobe analysis (EPMA). A broad range of Nb and Mo compositions were investigated for Si compositions up to 30 mol%. Microstructural and microchemical evidence provide a clear definition of the Nb–Mo–Si liquidus surface projection and an isothermal section diagram at 1973 K.

Experimental studies of light amplification with dynamic multiwave gratings recorded in an acceptor-donor-substituted azobenzene-doped poly(methyl methacrylate) thin film by multiwave mixing are reported. The amplification gain was measured to be as high as 6.7, and the operation at red light wavelength decreases the absorption losses.

To type Klebsiella pneumonia through methods including pulse-field gel electrophoresis (PFGE) in combination with multilocus sequence typing.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 typingBy 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.Highly similar Klebsiella pneumonias typed by PFGE could also be typed by MLST.

To investigate the serotype distribution and molecular features of Streptococcus pneumonia isolates in China.144 strains isolated from 6 provines were selected as the research object. The serotypes were determined with the method of capsular swelling. The molecular characteristics were conducted by optimized pulsed-field gel electrophoresis.A total of 135 Streptococcus pneumoniae isolates were divided into 14 serogroups. 19, 6, 15, 23 serogroups are prevalent types in China. 9 isolates were failed to identify specific serogroups. All isolates could be divided into 92 pulsotypes. Pulsotypes were scattered. Absolutely dominant pulsotype was not found. Pulsotypes were similar among same serogroup isolates.The pneumococcus of serogroup 19, 6, 15, 23 were common in China. PFGE had strong discriminatory ability of subtyping of Streptococcus pneumoniae. PFGE showed better epidemiological survey capacity.

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.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.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.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.

To investigate antibiotic resistance and the prevalence of serotype of Streptococcus pneumoniae.Totally 123 strains of S. pneumoniae were collected to test the susceptibility of 6 antibiotics by K-B method according to the standard of CLIS. Serotyping of S. pneumoniae was performed by using quelling reaction.Among 123 strains of S. pneumoniae, all strains (100%) were resistant to AZM. There were 65.04%, 78.05%, 19.51%, 0.81%, 0% of S. pneumoniae were resistant against the MH, SXT, C, RA, OFX respectively. 11 serotypes were involved in 123 strains. The prevalent serogroup were 6, 14, 15, 19 and 23.5 strains were unable to serotype. The multi-resistant strains of S. pneumoniae included in serogroup 6 and 19.The antibiotic resistance of S. pneumoniae is very serious. Most of them are multi-resistant strains. The prevalent serogroup, especially of the multi-resistant strains, are 6 and 19 serogroup.

To develop a rapid method for detecting 8 pathogens which were highly related to bacterial meningitis by multiplex polymerase chain reaction.By optimizing the reaction condition and amplification program of single pair polymerase chain reaction, the multiplex pairs polymerase chain reactions (M-PCR) was developed to identify eight pathogens simultaneously including Neisseria Meningitis, Haemophilus Influenzae, Streptococcus Pneumoniae, Cryptococcus Neoformans, Staphylococcus Aureus, Listerisa Monocytogene, Streptococcus Suis and Mycobacterium Tuberculosis. Meanwhile, The sensitivity of M-PCR assay was also studied.M-PCR methods for detecting 8 pathogens which could cause bacterial meningitis have been established. M-PCR showed specific, sensitive and more rapid than conventional culturing method.This multiplex polymerase chain reaction method can be used for diagnosis and scanning of suspicious bacterial meningitis cases in order to improve the diagnostic positive rate of bacterial meningitis cases.

To research the relationship between variable number of tandem repeats sequences (VNTRs) in genome and serogrouping of Neisseria meningitides (N. meningitidis).118 meningococcal strains which were isolated from invasive patients and carriers in China were serogrouped by antiseria agglutination testing. 38 strains were serogroup A. 80 were serogroup C. And then four variable number of tandem repeats sequences (VNTRs) loci in the DNA of 118 meningococcal strains were amplified by PCR. A dendrogram was constructed on the basis of DNA length of PCR productions by the Unweighted Pair Group Method with Arithmetic Averages (UPGMA). The two pair primers-specific were designed based on the nucleotide variant afterward the VNTR01 locus (T01-A and T01-AC, T01-C and T01-AC) to amplify 118 meningococcal strains.118 strains were classified into five VNTR groups. 98.3% (116/118) strains were identical between serogrouping and VNTRs genotyping. 97.3% (36/37) strains of serogroup A were categorized into group I of VNTRs genotyping. 98.7% (75/76) strains of serogroup C were categorized into group II of VNTRs genotyping. According to the presence or absence of PCR products using two pair primers-specific, 118 strains were classified into two groups which correspond to serogroup A and C. The sensitivity of primers-specific T01-A and T01-AC was 89.5% (34/38). The specificity of primers-specific T01-A and T01-AC was 100% (38/38). The sensitivity of primers-specific T01-C and T01-AC was 100% (80/80). The specificity of primers-specific T01-C and T01-AC was 95.2% (80/84).VNTRs genotyping has high relation to serogrouping A and C. The nucleotide sequence variations in the galactosyl-transferase gene related to the serogrouping A and C.

To establish TaqMan Real-Time PCR method for detection and identification of Neisseria meningitidis.Seven sets of primers and FAM-labeled probes targeting different genes of Neisseria meningitidis were designed and synthesized. ctrA gene was used for identification of N. meningitidis species. Six serogruops (A, B, C, X, Y, W135) of N. meningitidis were detected with following genes: sacB (A), siaD (B), siaD (C), xcbB (X), synF (Y) and synG (W135) respectively. Sensitivity and specificity of Real-Time PCR were assessed for different primers and probes. 121 cerebrospinal fluid (CSF) specimens from suspected N. meningitidis invasive meningitis cases were detected by latex agglutination test and Real-Time PCR assay simultaneously.79 N. meningitidis isolates of different serogroups could be detected and identified by seven sets of primers and probes in this study. Real-Time PCR seemed more sensitive than standard PCR by 10(1)-10(3) times. The respective sensitivities for ctrA, sacB, siaD (B), siaD (C), xcbB, synF and synG were 8, 8, 80, 8, 8, 80, 8 genome DNA copies in each reaction. Of the 121 CSF specimens, 11 were positive for Real-Time PCR and 6 for latex agglutination test.Real-Time PCR could rapidly detect and identify N. meningitidis of different serogroups and seemed more sensitive. It could be widely used for diagnose of invasive meningitis caused by N. meningitidis.

To study the characteristics of epidemiology and molecular typing on Neisseria meningitidis serogroup C strains associated with outbreaks of Anhui province and sporadic cases in China, using pulsed field gel electrophoresis (PFGE).212 Neisseria meningitidis serogroup C strains were isolated from invasive meningococcal cases, close contacts and healthy carriers, including 48 strains from Anhui province with 38 strains associated with serogroup C outbreaks. PFGE were performed by genomic DNA digestion with Nhe I restriction enzyme. The results of PFGE were analyzed by BioNumerics software (Version 4.0, Applied Maths BVBA, Belgium).A total number of 212 Neisseria meningitidis serogroup C isolates were typed by 43 patterns, named AH1 to AH43. In China, AH1 pattern was the major PFGE pattern with 69.3% (n = 147) of all strains, distributed in 11 provinces. Three types of PFGE patterns (AH1 to AH3) were found in 48 strains from Anhui province, in which, 93.8% (n = 45) belonged to AH1. 97.4% (n = 37) of 38 strains associated with serogroup C outbreaks in Anhui province showed AH1 pattern. A total of 53 serogroup C strains were isolated from invasive meningococcal cases with 67.9% (36/53) of AH pattern. 71.9% (87/121) of serogroup C strains isolated from contacts of invasive meningococcal cases was AH1 pattern and 63.2% (24/38) of the strains from healthy carriers showed AH1 pattern.By PFGE typing and analysis, AH1 pattern of Neisseria meningitidis serogroup C strains was proved to be the main clone which causing the outbreaks in Anhui province and might be responsible for the sporadic serogroup C meningococcal disease epidemics else where in the country.

Background Emergence of severe acute respiratory syndrome (SARS) from the winter of 2002 to the spring of 2003 has caused a serious threat to public health. Methods To evaluate the safety and immunogenicity of the inactivated SARS coronavirus (SARS-CoV) vaccine, 36 subjects received two doses of 16 SARS-CoV units (SU) or 32 SU inactivated SARS-CoV vaccine, or placebo control. Results On day 42, the seroconversion reached 100% for both vaccine groups. On day 56, 100% of participants in the group receiving 16 SU and 91.1% in the group receiving 32 SU had seroconverted. The geometric mean titre of neutralizing antibody peaked 2 weeks after the second vaccination, but decreased 4 weeks later. Conclusion The inactivated vaccine was safe and well tolerated and can elicit SARS-CoV-specific neutralizing antibodies.

To study the prevalence of severe acute respiratory syndrome coronavirus (SARS-CoV) like virus in animals at a live animal market of Guanzhou in 2004 before and after culling of wild animal action taken by the local authority, in order to predict the re-emerging of SARS from animal originals in this region.Animals at live animal market were sampled for rectal and throat swabs in triplicate. A single step realtime reverse transcription-polymerase chain reaction (RT-PCR) diagnostic kit was performed for screening SARS-CoV like virus, the manual nested RT- PCR and DNA sequencing were performed for confirmation. Only specimens which tested positive for both of the N and P genes by nested RT-PCR were scored as positive.In 31 animals sampled in January 5 2004 before culling of wild animals at Guangdong Province, including 20 cats (Felis catus), 5 red fox (Vulpes vulpes) and 6 Lesser rice field rats (Rattus losea), 8 (25.8%) animals were tested positive for SARS-CoV like virus by RT-PCR methods, of which 4 cats, 3 red fox and one Lesser rice field rats were included. However, two weeks after culling of animals and disinfection of the market were implemented, in 119 animals sampled in January 20 2004, including 6 rabbits (Oryctolagus cuniculus), 13 cats, 46 red jungle fowl (Gallus gallus), 13 spotbill duck (Anas platyrhynchos), 10 greylag goose (Anser anser), 31 Chinese francolin (Franclinus pintadeanus), only rectal swab from one greylag goose was tested positive for SARS-CoV like virus. Furthermore, in 102 animals that including 14 greylag gooses, 3 cats, 5 rabbits, 9 spotbill duck (Anaspoecilorhyncha), 2 Chinese francolin (Franclinus pintadeanus), 8 common pheasant (Phasianus colchicus), 6 pigeons, 9 Chinese muntjac (Muntiacus reevesi), 19 wild boar (Sus scrofa), 16 Lesser rice field rats, 5 dogs, 1 mink (Mustela vison), 3 goats, 2 green peafowl (Pavo muticus) sampled in April, May, June, July, August and November, only rectal swab from one pig was tested positive. However, of 12 and 10 palm civets sampled in November and December including five of which had been at the live animals market for 2 days, none of them was tested positive.This findings revealed that animals being sampled in April, May, June, July, August and November of 2004, only one rectal swab from a pig was tested positive as SARS-CoV like virus, much lower than the results from the previous year, suggesting that the possibility of re-emerging of human infection from animal origins is low for the winter of 2004-2005.