Development and implementation of standardized method for detecting immunogenicity of acellular pertussis vaccines in Korea

PURPOSE: There is no standard method for confirming the immunogenicity of acellular pertussis vaccines. We tried to develop a local standard method for evaluating the immunogenicity of the three-component of acellular pertussis vaccines which was developed by a Korean local company. MATERIALS AND METHODS: The developed pertussis antigens (pertussis toxin, filamentous hemagglutinin, pertactin) were evaluated by in-house enzyme-linked immunosorbent assay (ELISA) using 189 negative sera, 25 positive sera, and 73 paired sera (pre- and post-Tdap [tetanus, diphtheria, and acellular pertussis] vaccinated sera). ELISA units were calculated by the reference line method, compared with World Health Organization reference sera, and the cut-off value was calculated using negative sera. RESULTS: When compared to National Institute for Biological Standards and Control control antigen (NIBSC) control antigens, the developed pertussis toxin (PT) and filamentous haemagglutinin (FHA) antigens were 203.48 and 61.60 IU/µg, respectively. Each in-house ELISA was established by validating the coefficients of variation % (PT, 11.53%; FHA, 8.60%; pertactin [PRN], 9.86%) obtained from the results of inter- and intra-assay variation. Also, the cut-off values of PT, FHA, and PRN were 11.65, 38.95, and 5.66 EU/mL, respectively. The distributions of antibody levels in paired showed that 93.15% (68/73) in anti-PT IgG, 97.26% (72/73) in anti-FHA IgG, and 100% in anti-PRN IgG were higher than a 100% increase after vaccination. Additionally, the values of 89.04% (65/73) in anti-PT IgG, 97.26% (72/73) in anti-FHA IgG, and 100% in anti-PRN IgG were below each cut-off point. CONCLUSION: We established an in-house ELISA method using self-developed antigens, and these immunoassays have provided a way to standardize measuring the immunogenicity of newly developed vaccines, through single- and dual-serology.

Enzyme-linked immunosorbent assay for detecting anti-pertussis toxin antibody in mouse

PURPOSE: Although the DTaP and Tdap vaccines used to prevent pertussis have been used for a long time, there is no standard method for measuring pertussis antigens. Therefore, this preliminary study was conducted to develop an enzyme-linked immunosorbent assay method using an animal model for measuring antibodies against pertussis toxin, the most important pertussis pathogenic antigen, in the sera of vaccinated mice. MATERIALS AND METHODS: Bordetella pertussis Tohama phase I was cultured for 24–30 hours, and then pertussis toxin was purified from the culture medium by chromatography. Purified pertussis toxin was diluted in phosphate-buffered saline-coating buffer, and 100 µL of diluted pertussis toxin was added to each well and reacted at room temperature for 4 hours. Positive serum was diluted to 1/1,250–1/80,000 and negative serum was diluted to 1/50 to determine the coating concentration with the optimal signal/noise ratio. Optimal test conditions were confirmed from the dilution factors of the secondary antibody and streptavidin horseradish peroxidase (SA-HRP). RESULTS: Optimal conditions were as follows: 4 µg/mL for coating antigen; 1/40,000 for secondary antibody; and 1/1,000 for the SA-HRP dilution factor. Comparison of the sera obtained from mice treated with a developing vaccine and commercial vaccine with National Institute for Biological Standard and Control standard serum under the established conditions showed the following results: 1,300.62, 534.94, and 34.85, respectively. CONCLUSION: The method developed in this study is suitable for measuring anti-pertussis toxin antibodies and may be applicable for clinical sample analysis or indirect diagnosis of pertussis.

Preliminary study on the immunogenicity of a newly developed GCC Tdap vaccine and its protection efficacy against Bordetella pertussis in a murine intranasal challenge model

PURPOSE: Active reduced dose tetanus-diphtheria-acellular pertussis (Tdap) vaccination for adolescents and adults is necessary because waning immunity after primary diphtheria-tetanus-pertussis vaccination is related to the recent emergence of pertussis. This study was conducted to compare the immunogenicity and protection efficacy against Bordetella pertussis between a new GCC Tdap vaccine and a commercially available Tdap vaccine in a murine model. MATERIALS AND METHODS: BALB/c mice were immunized with two doses of diphtheria-tetanus-acellular pertussis (DTaP) vaccine for priming and a subsequent Tdap booster vaccination. According to the type of booster vaccine, mice were divided into four groups: commercially available Tdap vaccine in group 1 and GCC Tdap vaccines of different combinations of pertussis antigens in groups 2 to 4. Humoral and cell-mediated immune responses and protection efficacy using a murine intranasal challenge model after booster vaccination were compared among the four groups. RESULTS: Every group showed significant increases in antibody titers against pertussis antigens such as pertussis toxin, filamentous hemagglutinin, and pertactin after booster vaccination. Spleen cells showed both Th1 and Th2 cell-mediated immune responses stimulated by pertussis antigens in all groups without any significant difference. In the intranasal B. pertussis infection model, bacteria were eradicated in all groups five days after challenge infection. CONCLUSION: This preliminary study did not show significantly different immunogenicity or protection efficacy of the new GCC Tdap vaccines compared to the commercially available Tdap vaccine, although a more extensive study is necessary to assess the differing efficacies of the new GCC Tdap vaccines.

Antimicrobial Activities of Arbekacin against Clinical Isolates of Staphylococcus aureus and Coagulase-Negative Staphylococcus Species / 감염

BACKGROUND: Most strains of methicillin-resistant Staphylococcus aureus (MRSA) now exhibit high-level resistance to various antibiotics, such as beta-lactam antibiotics, aminoglycosides, macrolides, tetracyclines and quinolones. Recent reports describing the therapeutic failure of vancomycin for MRSA infections have arisen considerable concerns regarding the emergence of MRSA strains, which will require new therapeutic agents. Arbekacin, an aminoglycoside antibiotic, has antibacterial activity against both gram-positive and gram-negative bacteria and is stable in the presence of aminoglycoside inactivating enzymes produced by S. aureus. In this study, we compared the antibacterial activity of arbekacin with those of vancomycin, gentamicin, and amikacin against Staphylococcus aureus (S. aureus) and coagulase-negative staphylococci (CNS). METHODS: For a collection of 549 S. aureus and 251 CNS isolates from three Catholic University Hospitals in Korea, minimum inhibitory concentrations (MICs) of arbekacin, vancomycin, amikacin and gentamicin were determined by agar dilution method using Mueller-Hinton agar according to NCCLS (National Committee for Clinical Laboratory Standards, USA) criteria. RESULTS: Among 549 S. aureus isolates, 278 isolates were MRSA and 271 isolates were methicillin- sensitive S. aureus (MSSA). MIC50 & MIC90 of arbekacin against 549 S. aureus were 0.5 & 1 microgram/mL, and MIC50 & MIC90 of vancomycin were 1 & 1 microgram/ mL. MIC of arbekacin against 549 S. aureus isolates ranges from 0.03 to 4 microgram/mL, and MIC of vancomycin against 549 S. aureus ranges from 0.25 to 2 microgram/ mL. MIC90 of amikacin against 549 S. aureus was 32 microgram/mL, and that of gentamicin was 128 microgram/mL. MICs of amikacin and gentamicin were variable, ranging from 0.125 to 256, and otherwise arbekacin and vancomycin revealed relatively narrow range of MICs. MIC90 of arbekacin against 278 MRSA isolates & 271 MSSA were 1 & 0.5 microgram/mL, and those of vancomycin against MRSA & MSSA were 1 & 1 microgram/mL. MIC90 of amikacin against 278 MRSA & 271 MSSA isolates were 32 & 4 microgram/mL, and that of gentamicin against MRSA & MSSA isolates were 128 & 32 microgram/ mL respectively. Among 251 CNS isolates, 122 isolates were MRCNS and 129 were MSCNS. MIC50 & MIC90 of arbekacin against 251 CNS isolates were 0.25 & 2 microgram/mL, and those of vancomycin were 1 & 2 microgram/mL. MIC of arbekacin against 251 CNS isolates ranges from 0.015 to 32 microgram/mL, and that of vancomycin isolates ranges from 0.25 to 2 microgram/mL. MIC90 of arbekacin against 122 MRCNS & 129 MSCNS isolates were 2 & 0.5 microgram/mL, and those of vancomycin were 2 & 2 microgram/mL. MIC90 of amikacin against 251 CNS isolates was 32 microgram/mL, and that of gentamicin was 128 microgram/mL for CNS. MIC90 of amikacin against 122 MRCNS & 129 MSCNS isolates were 128 & 8 microgram/mL, and those of gentamicin were 256 & 32 microgram/ mL. CONCLUSION: Considering above results, arbekacin can be a useful agent against most strains of MRSA and MRCNS, which exhibit high-level resistance to amikacin and gentamicin.

Clinical Characteristics of Herpes Zoster in Immunocompromised Patients and Analysis of Pharmacokinetics of Acyclovir / 감염

BACKGROUND: We investigated this study to elucidate the clinical characteristics of herpes zoster in immunocompromised patients and to analyze the pharmacokinetics of acyclovir with the response of therapy. METHODS: A total of 51 immunocompromised patients with herpes zoster were studied prospectively over 22 months (Dec. 1997-Sep. 1999). Patients were randomized to 4 groups according to pharmaceutical company (company A or B) and method of infusion (intermittently or continuously) of acyclovir. Patients were assigned to receive acyclovir (10 mg/kg, three times daily) intermittently, or acyclovir (5 mg/kg bolus, and then 40 mg/kg/day) continuously for 7 days respectively. RESULTS: Mean age was 31.9+/-12.6 years and the ratio of male to female was 1:1.68. Dermatome involvement was most frequently on the thoracic dermatome (49%), followed by cervical, lumbar dermatome. Forty-two (82.3%) patients received hematopoietic stem cell transplantation and herpes zoster was most prevalent in average 9.2+/-7.9 months after transplantation. Thirty (58%) patients had been taken immunosuppressants at the onset of herpes zoster. Recurrence rate of herpes zoster was 7.8%. Overall adverse experience rate was 15.7%. Pharmacokinetic parameter of acyclovir from company B was close to reference as compared with those of company A. There was no difference in steady-state concentration (Css) of acyclovir between intermittent and continuous infusion. Cessation of new lesion formation occurred 4.1+/-1.3 days after initiation of therapy without statistically significant intergroup differences. Rate to loss of vesicle over 50% at the seventh day of infusion also showed no intergroup differences, but tended to highest at the continuous group of company B. CONCLUSION: Herpes zoster in immunocompromised patients were prevalent during the use of immunosuppressant, mostly within 1 year after hematopoietic stem cell transplantation. Anatomical distribution was just like that of immunocompetent patients, but recurred more frequently. Clinical response was not different according to the pharmaceutical company or method of infusion. Supplementary evaluation to the dose of acyclovir, method of infusion, duration of treatment, and alternatives may be required.

In vitro activities of quinupristin/dalfopristin and eight other antimicrobial agents against 360 clinical isolates from Korea

The emergence of multi-drug resistant gram-positive cocci such as methicillin-resistant (MR) staphylococci, vancomycin-resistant (VR) enterococci, and vancomycin-intermediate resistant S. aureus (VISA) has given new urgency to the development of new antimicrobial agents. One of these is quinupristin/dalfopristin (Q/D). We decided to determine the susceptibility of gram-positive cocci isolated at two university hospitals in Seoul to Q/D and compare the results with eight other antimicrobial agents. We investigated 120 isolates of S. aureus including 49 MRSAs and one VISA, 120 isolates of coagulase negative staphylococci (CNS), 64 E. faecalis and 56 E. faecium, including seven strains of VR E. faecium. Minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) for several antimicrobials, including vancomycin and Q/D, were determined by broth microdilution. All S. aureus including VISA were susceptible to Q/D. Q/D MIC90 for both methicillin-susceptible S. aureus (MSSA) and MRSA was 0.25 g/mL. 49 (87.5%) of 56 E. faecium including six of seven VR E. faecium were susceptible to Q/D. E. faecalis were not susceptible to Q/D (only 1.5% susceptible), but were inhibited by ampicillin (94% susceptible) or vancomycin (95%). CNS was susceptible to Q/D (96% susceptible) and vancomycin (100% susceptible). One of 38 staphylococci and two of 17 E. faecium were tolerant to Q/D. In conclusion, Q/D showed excellent activity against all species of gram-positive cocci including MRSA, VISA, and VR E. faecium except E. faecalis, and may provide a valuable option for the treatment of infections caused by these emerging nosocomial pathogens of gram-positive cocci.

Apoptosis of Peripheral Blood Cells Stimulated by Lipopolysaccharide, Cytokines, or Escherichia coli / 감염

INTRODUCTION AND METHODS: Apoptosis is a natural suicidal mechanism of eukaryotic cells to maintain the homeostasis of hosts. It comprises important pathogenesis of autoimmune diseases, cancers, and some infectious diseases. To access the role of apoptosis in the pathogenesis of infectious diseases, we studied the difference in apoptotic patterns of polymorphonuclear leukocytes and of monocytes to various stimuli, such as lipopolysaccharide (LPS), tumor necrosis factor (TNF)-alpa, interleukin (IL)-6, granulocyte, macrophage-colony stimulating factor (GM-CSF), or Escherichia coli (E. coli). The cell survival and expression of Fas and Fas ligand (FasL), bcl-2, and IL-1beta converting enzyme (ICE) were evaluated by flow cytometry, northern blotting or western blotting analysis. RESULTS: The survival of polymorphonuclear leukocytes and monocytes was prolonged after stimulation with LPS and cytokines. Stimulation of Fas with Fas monoclonal antibody induced apoptosis in both polymorphonuclear leukocytes and monocytes. Expressions of Fas and FasL were more dominant in polymorphonuclear leukocytes than in monocytes. bcl-2 was expressed onlyin monocytes and was associated with the de-lay of apoptosis. ICE is a common pathway to apoptosis and thus pretreatment with ICE inhibitor could partially inhibit the apoptosis in both cells. Interestingly, E. coli prolonged the life-span of polymorphonuclear leukocytes but accelerated apoptosis in monocytes in spite of the over-expression of bcl-2. CONCLUSION: Polymorphonuclear leukocytes and monocytes might have different apoptotic mechanisms to maintain their homeostasis. Polymorphonuclear leukocytes have only one mechanism to apoptosis, namely via Fas- FasL, whereas, monocytes have both Fas-FasL and bcl-2 mechanisms. These differences may be associated with their primary functions and natural life-span. Direct stimulation by live E. coli accelerated apoptosis in monocytes in spite of the over-expression of inhibitor of apoptosis, i.e. bcl-2. It could be due to activation of other apoptotic mechanisms by E. coli, which may detour the anti-apoptotic action of bcl-2 or direct inhibition of bcl-2 function.

Molecular analysis of fluoroquinolone-resistance in Escherichia coli on the aspect of gyrase and multiple antibiotic resistance (mar) genes

We analyzed the fluoroquinolone resistance mechanism of 28 isolates of ciprofloxacin-resistant E. coli from patients who received ciprofloxacin as a regimen of a selective gut decontamination. Isolates distinctive by infrequent restriction site polymerase chain reaction (IRS-PCR) were subjected to Hinf I restriction fragment length polymorphism analysis, single-stranded conformation polymorphism (SSCP), and nucleotide sequencing of the quinolone resistance determining region (QRDR) in gyrA. Double mutations in QRDR of gyrA (Ser83 Leu and Asp87Asn) were found from most of the strains. Nucleotide sequencing of the marR locus showed that 18 out of 28 (64%) ciprofloxacin-resistant E. coli strains had three types of base change in marR loci: a double-base change at nucleotides 1628 and 1751, or 1629 and 1751: and a single-base change at 1751. However, all the mutated strains showed no tolerance to cyclohexane test, suggesting the mutation in the marR region had no influence on overexpression of the MarA protein. In conclusion, mutation in gyrA was the main mechanism of ciporfloxacin resistance in E. coli from patients with selective gut decontamination. Therefore, mutation in the mar region did not influence the levels of ciprofloxacin resistance in our isolates.

Species Identification of Enterococcus by Ribotyping / 감염

BACKGROUND: Emerging drug resistance and increasing incidence of enterococci infection necessitates an accurate species identification. METHOD: We compared the identification of twelve strains of American Type Culture Coliection(ATCC) (E. faecalis, E. avium, E. faecium, E. raffinosus, E. durans, E. casseliflavus, E. hirae, E. flavescens, E. malodoratus, E. gallnarum, E. mundtii, and E. sulfureus) and 73 clinical isolates of enterococci(E. faecalis 8, E. faecium 8, E. avium 13, E. durans 5, and unidentified strains 39) by Vitek Gram-positive Identification card (version R08.1, bio- Merieux Vitek, Hazelwood, Mo., U.S.A.), ribotyping and conventional scheme of Facklam and Sahm. RESULTS: All ATCC strains could be identified on the basis of characteristic 16S-23S rDNA fingerprint patterns by using Eel I and Hind III. Of 39 strains unidentified by Vitek GPI card, 25 strains were identified as E. faecium, 5 strains were E. gallinarum, 5 strains were E. casseliflavus, 3 strains were E. avium and 1 strain was E. hirae. CONCLUSION: By ribotyping with Eel I and Hind m digestion, all twelve ATCC strains and 73 clinical strains were identified and it seems to be a reliable method for identification of Enterococcus species.