We tested correlations between anti-Helicobacter pylori IgG and IgA levels and the urease test, anti-CagA protein antibody, degree of gastritis, and age. In total, 509 children (0-15 years) were enrolled. Subjects were stratified as 0-4 years (n = 132), 5-9 years (n = 274), and 10-15 years (n = 103) and subjected to the urease test, histopathology, ELISA, and western blot using whole-cell lysates of H. pylori strain 51. The positivity rate in the urease test (P = 0.003), the degree of chronic gastritis (P = 0.021), and H. pylori infiltration (P < 0.001) increased with age. The median titer for anti-H. pylori IgG was 732.5 IU/mL at 0-4 years, 689.0 IU/mL at 5-9 years, and 966.0 IU/mL at 10-15 years (P < 0.001); the median titer for anti-H. pylori IgA was 61.0 IU/mL at 0-4 years, 63.5 IU/mL at 5-9 years, and 75.0 IU/mL at 10-15 years (P < 0.001). The CagA-positivity rate was 26.5% at 0-4 years, 36.5% at 5-9 years, and 46.6% at 10-15 years for IgG (P = 0.036), and 11.3% at 0-4 years, 18.6% at 5-9 years, and 23.3% at 10-15 years for IgA (P < 0.001). Anti-H. pylori IgG and IgA titers increased with the urease test grade, chronic gastritis degree, active gastritis, and H. pylori infiltration. Presence of CagA-positivity is well correlated with a high urease test grade and high anti-H. pylori IgG/IgA levels.
Adolescent ; Antibodies, Bacterial/*blood ; Antigens, Bacterial/*analysis/immunology ; Bacterial Proteins/*analysis/immunology/metabolism ; Blotting, Western ; Child ; Child, Preschool ; Chronic Disease ; Enzyme-Linked Immunosorbent Assay ; Female ; Gastritis/pathology ; Helicobacter Infections/blood/microbiology/*pathology ; Helicobacter pylori/isolation & purification/*metabolism ; Humans ; Immunoglobulin A/*blood ; Immunoglobulin G/*blood ; Infant ; Infant, Newborn ; Male ; Severity of Illness Index ; Urease/metabolism

PURPOSE: Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. MATERIALS AND METHODS: The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. RESULTS: The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7x10(4) CFU/mL and 2.0x10(6) CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). CONCLUSION: The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.
Antigens, Bacterial/*analysis/immunology ; Enzyme-Linked Immunosorbent Assay/*methods ; Mycobacterium tuberculosis/*immunology

Pertussis is a representative vaccine-preventable disease. However, there have been recent outbreaks in countries where even higher vaccination against the disease. One reason is the emergence of antigenic variants, which are different to vaccine type. In Korea, reported cases have rapidly increased since 2009. Therefore, we analyzed genotype of strains isolated in 2011-2012 by multilocus sequence typing method. As expected, the genotype profiles of tested genes dramatically changed. The major sequence type changed from ST1 to ST2, and new sequence type (ST8) appeared. In the minimum spanning tree, recent isolates belonging to the ACC-I-ST3 subgroup were detected that were composed of ST2, ST3, and ST6. In particular, the ST2 frequency increased to 81%. The novel ST8 was linked to the increased frequency of ST2. In addition, toxic strains carrying the ptxP3 promoter type were confirmed. This ptxP3 type emerged from 2009 and its frequency had increased to 100% in 2012. Based on these results, it can be inferred that the genotypic changes in the currently circulating strains are strongly associated with the recent increasing of pertussis in Korea. Therefore, the surveillance system should be strengthened, and genetic characterization of the isolates should be expanded to the whole genome sequence level.
*Antigenic Variation ; Antigens/*genetics/immunology/metabolism ; Bacterial Proteins/genetics/metabolism ; Bordetella pertussis/*genetics/isolation & purification/*metabolism ; Genes, Bacterial ; Genotype ; Humans ; Pertussis Toxin/genetics/metabolism ; Promoter Regions, Genetic ; Republic of Korea ; Sequence Analysis, DNA ; Whooping Cough/immunology/*microbiology/pathology

There are limited data evaluating the relationship between influenza treatment and hospitalization duration. Our purpose assessed the association between different treatments and hospital stay among Korean pediatric influenza patients. Total 770 children < or = 15 yr-of-age hospitalized with community-acquired laboratory-confirmed influenza at three large urban tertiary care hospitals were identified through a retrospective medical chart review. Demographic, clinical, and cost data were extracted and a multivariable linear regression model was used to assess the associations between influenza treatment types and hospital stay. Overall, there were 81% of the patients hospitalized with laboratory-confirmed influenza who received antibiotic monotherapy whereas only 4% of the patients received oseltamivir monotherapy. The mean treatment-related charges for hospitalizations treated with antibiotics, alone or with oseltamivir, were significantly higher than those treated with oseltamivir-only (P < 0.001). Influenza patients treated with antibiotics-only and antibiotics/oseltamivir combination therapy showed 44.9% and 28.2%, respectively, longer duration of hospitalization compared to those treated with oseltamivir-only. Patients treated with antibiotics, alone or combined with oseltamivir, were associated with longer hospitalization and significantly higher medical charges, compared to patients treated with oseltamivir alone. In Korea, there is a need for more judicious use of antibiotics, appropriate use of influenza rapid testing.
Adolescent ; Anti-Bacterial Agents/*therapeutic use ; Antigens, Viral/analysis/immunology ; Antiviral Agents/*therapeutic use ; Child ; Child, Preschool ; Cohort Studies ; Demography ; Drug Therapy, Combination ; Female ; Hospitalization ; Humans ; Infant ; Infant, Newborn ; Influenza A virus/metabolism ; Influenza B virus/metabolism ; Influenza, Human/*drug therapy ; Male ; Oseltamivir/*therapeutic use ; Republic of Korea ; Retrospective Studies

OBJECTIVELcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study.

METHODSA new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography.

RESULTSRemoval of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)₃ adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10⁶ CFU of Y. pestis virulent strain 141.

CONCLUSIONThe completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

Amino Acid Sequence ; Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; genetics ; immunology ; Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Female ; Genetic Vectors ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Plague ; immunology ; prevention & control ; Plague Vaccine ; genetics ; immunology ; Plasmids ; Pore Forming Cytotoxic Proteins ; genetics ; immunology ; Protein Engineering ; methods ; Recombinant Fusion Proteins ; genetics ; immunology ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Survival Analysis ; Vaccines, Subunit ; genetics ; immunology ; Yersinia pestis ; growth & development ; immunology

The present study was conducted to examine the morphology and antigenicity of Photobacterium damselae subsp. piscicida by culturing the bacterium in vivo in the peritoneal cavity of sea bass (Dicentrarchus labrax) within dialysis bags with either a low molecular weight (LMW) cut-off of 25 kDa or a high molecular weight (HMW) cut-off of 300 kDa. Differences were observed in the growth rate between the bacteria cultured in vivo or in vitro. Bacteria cultured in vivo were smaller and produced a capsular layer, which was more prominent in bacteria cultured in the HMW bag. Antigenicity was examined by Western blot analysis using sera from sea bass injected with live Ph. d. subsp. piscicida. The sera recognised bands at 45 and 20 kDa in bacteria cultured in vivo in the LMW bag. Bacteria cultured in vivo in the HMW bag did not express the 45 kDa band when whole cell extracts were examined, although the antigen was present in their extracellular products. In addition, these bacteria had a band at 18 kDa rather than 20 kDa. Differences in glycoprotein were also evident between bacteria cultured in vitro and in vivo. Bacteria cultured in vitro in LMW and HMW bags displayed a single 26 kDa band. Bacteria cultured in the LMW bag in vivo displayed bands at 26 and 27 kDa, while bacteria cultured in vivo in the HMW bag possessed only the 27 kDa band. These bands may represent sialic acid. The significance of the changes observed in the bacterium's structure and antigenicity when cultured in vivo is discussed.
Animals ; Antigenic Variation/*genetics ; Antigens, Bacterial/genetics/*immunology ; Bass/*immunology/microbiology ; Blotting, Western ; Carbohydrates/analysis ; Electrophoresis, Polyacrylamide Gel ; Membranes, Artificial ; Microscopy, Electron, Transmission ; N-Acetylneuraminic Acid/genetics/*immunology ; Photobacterium/genetics/*immunology/ultrastructure

OBJECTIVETo reconstruct the nucleotide sequence of Leptospira interrogans lipL21 gene for increasing the output of prokaryotic expression and to understand the changes on immunogenicity of the expression products before and after reconstruction, and to determine the position of envelope lipoprotein LipL21 on the surface of leptospiral body.

METHODSAccording to the preferred codons of E.coli, the nucleotide sequence of lipL21 gene was designed and synthesized, and then its prokaryotic expression system was constructed. By using SDS-PAGE plus BioRad agarose image analysor, the expression level changes of lipL21 genes before and after reconstruction were measured. A Western blot assay using rabbit anti-TR/Patoc I serum as the first antibody was performed to identify the immunoreactivity of the two target recombinant proteins rLipL21s before and after reconstruction. The changes of cross agglutination titers of antisera against two rLipL21s before and after reconstruction to the different leptospiral serogroups were demonstrated using microscope agglutination test (MAT). Immuno-electronmicroscopy was applied to confirm the location of LipL21s.

RESULTThe expression outputs of original and reconstructed lipL21 genes were 8.5 % and 46.5 % of the total bacterial proteins, respectively. Both the two rLipL21s could take place immune conjugation reaction with TR/Patoc I antiserum. After immunization with each of the two rLipL21s in rabbits, the animals could produce specific antibody. Similar MAT titers with 1:80 - 1:320 of the two antisera against rLipL21s were present. LipL21 was confirmed to locate on the surface of leptospiral envelope.

CONCLUSIONLipL21 is a superficial antigen of Leptospira interrogans. The expression output of the reconstructed lipL21 gene is remarkably increased. The expression rLipL21 maintains fine antigenicity and immunoreactivity and its antibody still shows an extensive cross immunoagglutination activity. The high expression of the reconstructed lipL21 gene will offer a favorable condition to use its product for further developing a novel universal vaccine as well as detection kit of leptospirosis.

Amino Acid Sequence ; Animals ; Antigens, Bacterial ; genetics ; immunology ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; metabolism ; Bacterial Vaccines ; immunology ; Base Sequence ; Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Immune Sera ; immunology ; Leptospira interrogans ; genetics ; immunology ; ultrastructure ; Lipoproteins ; genetics ; immunology ; metabolism ; Microscopy, Immunoelectron ; Molecular Sequence Data ; Rabbits ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Sequence Analysis, DNA ; Vaccines, DNA ; immunology

aboratory diagnosis of tuberculosis (TB) traditionally relies on smear microscopy and culture of Mycobacterium tuberculosis from clinical samples. With recent advances in technology, there have been numerous efforts to develop new diagnostic tests for TB that overcome the low sensitivity and specificity and long turnover time associated with current diagnostic tests. Molecular biological tests based on nucleic acid amplification have brought an unprecedented opportunity for the rapid and specific detection of M. tuberculosis from clinical specimens. With automated sequencing analysis, species identification of mycobacteria is now easier and more accurate than with conventional methods, and rapid detection of mutations in the genes associated with resistance to TB drugs provides early information on the potential drug resistance for each clinical isolate or for clinical samples. In addition, immunological tests for the detection of M. tuberculosis antigens and antibodies to the antigens have been explored to identify individuals at risk of developing TB or with latent TB infection (LTBI). The recent introduction of commercial IFN-gamma assay kits for the detection of LTBI provides a new approach for TB control even in areas with a high incidence of TB. However, these molecular and immunological tools still require further evaluation using large scale cohort studies before implementation in TB control programs.
Antigens, Bacterial/immunology ; DNA, Bacterial/chemistry/genetics ; Humans ; Immunologic Tests/*methods ; Interferon-gamma/analysis ; Mycobacterium tuberculosis/genetics/immunology ; Sequence Analysis, DNA ; Tuberculin Test ; Tuberculosis/*diagnosis/immunology/microbiology

aboratory diagnosis of tuberculosis (TB) traditionally relies on smear microscopy and culture of Mycobacterium tuberculosis from clinical samples. With recent advances in technology, there have been numerous efforts to develop new diagnostic tests for TB that overcome the low sensitivity and specificity and long turnover time associated with current diagnostic tests. Molecular biological tests based on nucleic acid amplification have brought an unprecedented opportunity for the rapid and specific detection of M. tuberculosis from clinical specimens. With automated sequencing analysis, species identification of mycobacteria is now easier and more accurate than with conventional methods, and rapid detection of mutations in the genes associated with resistance to TB drugs provides early information on the potential drug resistance for each clinical isolate or for clinical samples. In addition, immunological tests for the detection of M. tuberculosis antigens and antibodies to the antigens have been explored to identify individuals at risk of developing TB or with latent TB infection (LTBI). The recent introduction of commercial IFN-gamma assay kits for the detection of LTBI provides a new approach for TB control even in areas with a high incidence of TB. However, these molecular and immunological tools still require further evaluation using large scale cohort studies before implementation in TB control programs.
Antigens, Bacterial/immunology ; DNA, Bacterial/chemistry/genetics ; Humans ; Immunologic Tests/*methods ; Interferon-gamma/analysis ; Mycobacterium tuberculosis/genetics/immunology ; Sequence Analysis, DNA ; Tuberculin Test ; Tuberculosis/*diagnosis/immunology/microbiology

No abstract available.
Adolescent ; Adult ; Antigens, Bacterial/analysis ; Chlamydia Infections/*diagnosis/epidemiology/microbiology ; Chlamydia trachomatis/immunology ; DNA, Viral/analysis ; Female ; Humans ; Mass Screening/*methods ; Papillomavirus Infections/*diagnosis/epidemiology/virology ; Papillomavirus, Human/genetics ; Poland/epidemiology ; Prevalence ; *Sexual Behavior ; Urban Population