PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-20 (20)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
more »
1.  A Three-Dose Intramuscular Injection Schedule of Anthrax Vaccine Adsorbed Generates Sustained Humoral and Cellular Immune Responses to Protective Antigen and Provides Long-Term Protection against Inhalation Anthrax in Rhesus Macaques 
Clinical and Vaccine Immunology : CVI  2012;19(11):1730-1745.
A 3-dose (0, 1, and 6 months) intramuscular (3-IM) priming series of a human dose (HuAVA) and dilutions of up to 1:10 of anthrax vaccine adsorbed (AVA) provided statistically significant levels of protection (60 to 100%) against inhalation anthrax for up to 4 years in rhesus macaques. Serum anti-protective antigen (anti-PA) IgG and lethal toxin neutralization activity (TNA) were detectable following a single injection of HuAVA or 1:5 AVA or following two injections of diluted vaccine (1:10, 1:20, or 1:40 AVA). Anti-PA and TNA were highly correlated (overall r2 = 0.89 for log10-transformed data). Peak responses were seen at 6.5 months. In general, with the exception of animals receiving 1:40 AVA, serum anti-PA and TNA responses remained significantly above control levels at 28.5 months (the last time point measured for 1:20 AVA), and through 50.5 months for the HuAVA and 1:5 and 1:10 AVA groups (P < 0.05). PA-specific gamma interferon (IFN-γ) and interleukin-4 (IL-4) CD4+ cell frequencies and T cell stimulation indices were sustained through 50.5 months (the last time point measured). PA-specific memory B cell frequencies were highly variable but, in general, were detectable in peripheral blood mononuclear cells (PBMC) by 2 months, were significantly above control levels by 7 months, and remained detectable in the HuAVA and 1:5 and 1:20 AVA groups through 42 months (the last time point measured). HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming, with sustained production of high-avidity PA-specific functional antibody, long-term immune cell competence, and immunological memory (30 months for 1:20 AVA and 52 months for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high-magnitude anamnestic anti-PA IgG and TNA responses.
doi:10.1128/CVI.00324-12
PMCID: PMC3491539  PMID: 22933399
2.  Accuracy of real-time PCR, Gram stain and culture for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae meningitis diagnosis 
Background
Although cerebrospinal fluid (CSF) culture is the diagnostic reference standard for bacterial meningitis, its sensitivity is limited, particularly when antibiotics were previously administered. CSF Gram staining and real-time PCR are theoretically less affected by antibiotics; however, it is difficult to evaluate these tests with an imperfect reference standard.
Methods and findings
CSF from patients with suspected meningitis from Salvador, Brazil were tested with culture, Gram stain, and real-time PCR using S. pneumoniae, N. meningitidis, and H. influenzae specific primers and probes. An antibiotic detection disk bioassay was used to test for the presence of antibiotic activity in CSF. The diagnostic accuracy of tests were evaluated using multiple methods, including direct evaluation of Gram stain and real-time PCR against CSF culture, evaluation of real-time PCR against a composite reference standard, and latent class analysis modeling to evaluate all three tests simultaneously.
Results
Among 451 CSF specimens, 80 (17.7%) had culture isolation of one of the three pathogens (40 S. pneumoniae, 36 N. meningitidis, and 4 H. influenzae), and 113 (25.1%) were real-time PCR positive (51 S. pneumoniae, 57 N. meningitidis, and 5 H. influenzae). Compared to culture, real-time PCR sensitivity and specificity were 95.0% and 90.0%, respectively. In a latent class analysis model, the sensitivity and specificity estimates were: culture, 81.3% and 99.7%; Gram stain, 98.2% and 98.7%; and real-time PCR, 95.7% and 94.3%, respectively. Gram stain and real-time PCR sensitivity did not change significantly when there was antibiotic activity in the CSF.
Conclusion
Real-time PCR and Gram stain were highly accurate in diagnosing meningitis caused by S. pneumoniae, N. meningitidis, and H. influenzae, though there were few cases of H. influenzae. Furthermore, real-time PCR and Gram staining were less affected by antibiotic presence and might be useful when antibiotics were previously administered. Gram staining, which is inexpensive and commonly available, should be encouraged in all clinical settings.
doi:10.1186/1471-2334-13-26
PMCID: PMC3558362  PMID: 23339355
Bacterial meningitis; Diagnostic test evaluation; Real-time PCR; Streptococcus pneumoniae; Neisseria meningitidis; Haemophilus influenzae
3.  Interlaboratory Standardization of the Sandwich Enzyme-Linked Immunosorbent Assay Designed for MATS, a Rapid, Reproducible Method for Estimating the Strain Coverage of Investigational Vaccines 
Clinical and Vaccine Immunology : CVI  2012;19(10):1609-1617.
The meningococcal antigen typing system (MATS) sandwich enzyme-linked immunosorbent assay (ELISA) was designed to measure the immunologic cross-reactivity and quantity of antigens in target strains of a pathogen. It was first used to measure the factor H-binding protein (fHbp), neisserial adhesin A (NadA), and neisserial heparin-binding antigen (NHBA) content of serogroup B meningococcal (MenB) isolates relative to a reference strain, or “relative potency” (RP). With the PorA genotype, the RPs were then used to assess strain coverage by 4CMenB, a multicomponent MenB vaccine. In preliminary studies, MATS accurately predicted killing in the serum bactericidal assay using human complement, an accepted correlate of protection for meningococcal vaccines. A study across seven laboratories assessed the reproducibility of RPs for fHbp, NadA, and NHBA and established qualification parameters for new laboratories. RPs were determined in replicate for 17 MenB reference strains at laboratories A to G. The reproducibility of RPs among laboratories and against consensus values across laboratories was evaluated using a mixed-model analysis of variance. Interlaboratory agreement was very good; the Pearson correlation coefficients, coefficients of accuracy, and concordance correlation coefficients exceeded 99%. The summary measures of reproducibility, expressed as between-laboratory coefficients of variation, were 7.85% (fHbp), 16.51% (NadA), and 12.60% (NHBA). The overall within-laboratory measures of variation adjusted for strain and laboratory were 19.8% (fHbp), 28.8% (NHBA), and 38.3% (NadA). The MATS ELISA was successfully transferred to six laboratories, and a further laboratory was successfully qualified.
doi:10.1128/CVI.00202-12
PMCID: PMC3485892  PMID: 22875603
4.  Comparative Study of Different Sources of Pertussis Toxin (PT) as Coating Antigens in IgG Anti-PT Enzyme-Linked Immunosorbent Assays 
In an effort to improve the reliability and reproducibility of serological assays for Bordetella pertussis, a collaborative study was conducted to compare four different sources of pertussis toxin (PT) as coating antigens in the immunoglobulin G (IgG) anti-PT enzyme-linked immunosorbent assay (ELISA). Four sources of PT were used as coating antigens in the IgG anti-PT ELISA in four different testing laboratories (labs A to D) to determine whether the different antigen preparations and different laboratories influenced assay results. A panel of 60 sera consisting of deidentified human specimens from previous vaccination trials of healthy adults and infants and clinical specimens from outbreak settings was tested. In the four laboratories, each sample was tested three times with the four PT antigens according to the standard coating optimization and IgG anti-PT ELISA testing procedures used in that laboratory. Differences among the antigens, as well as intra- and interlaboratory variability, were evaluated. Excellent agreement was observed with the test panel results among the four antigens within each laboratory. Concordance correlation coefficient (rc) measurements among the different antigens ranged from 0.99, 0.99 to 1.00, 1.00, and 0.97 to 1.00 for labs A to D, respectively. The comparisons between pairs of laboratories also indicated a high degree of concordance for each PT preparation, with rc measurements between 0.90 and 0.98, 0.93 and 0.99, 0.92 and 0.98, and 0.93 and 0.99 for antigens 1 to 4, respectively. Relatively minor differences in results were observed among laboratories or among antigens, suggesting that the four PT antigens are quite similar and could be considered for acceptance in harmonized immunoassays used for serodiagnosis or vaccine evaluation.
doi:10.1128/CVI.05460-11
PMCID: PMC3255951  PMID: 22116688
5.  Multilaboratory Comparison of Streptococcus pneumoniae Opsonophagocytic Killing Assays and Their Level of Agreement for the Determination of Functional Antibody Activity in Human Reference Sera ▿ †  
Antibody-mediated killing of Streptococcus pneumoniae (pneumococcus) by phagocytes is an important mechanism of protection of the human host against pneumococcal infections. Measurement of opsonophagocytic antibodies by use of a standardized opsonophagocytic assay (OPA) is important for the evaluation of candidate vaccines and required for the licensure of new pneumococcal conjugate vaccine formulations. We assessed agreement among six laboratories that used their own optimized OPAs on a panel of 16 human reference sera for 13 pneumococcal serotypes. Consensus titers, estimated using an analysis-of-variance (ANOVA) mixed-effects model, provided a common reference for assessing agreement among these laboratories. Agreement was evaluated in terms of assay accuracy, reproducibility, repeatability, precision, and bias. We also reviewed four acceptance criterion intervals for assessing the comparability of protocols when assaying the same reference sera. The precision, accuracy, and concordance results among laboratories and the consensus titers revealed acceptable agreement. The results of this study indicate that the bioassays evaluated in this study are robust, and the resultant OPA values are reproducible for the determination of functional antibody titers specific to 13 pneumococcal serotypes when performed by laboratories using highly standardized but not identical assays. The statistical methodologies employed in this study may serve as a template for evaluating future multilaboratory studies.
doi:10.1128/CVI.00370-10
PMCID: PMC3019776  PMID: 21084458
6.  Interlaboratory Comparison of Three Multiplexed Bead-Based Immunoassays for Measuring Serum Antibodies to Pneumococcal Polysaccharides ▿  
Serotype-specific IgG, as quantified by a standardized WHO enzyme-linked immunosorbent assay (ELISA), is a serologic end point used to evaluate pneumococcal polysaccharide-based vaccine immunogenicity. Antibodies to each vaccine polysaccharide in licensed multivalent vaccines are quantified separately; this is laborious and consumes serum. We compared three bead-based immunoassays: a commercial assay (xMAP Pneumo14; Luminex) and two in-house assays (of the Health Protection Agency [HPA] and Centers for Disease Control and Prevention [CDC]), using the WHO-recommended standard reference and reference sera (n = 11) from vaccinated adults. Multiple comparisons of the IgG concentrations for seven conjugate vaccine serotypes were performed by sample (percent error), serotype (equivalency testing), and laboratory (concordance correlation coefficient [CCC]). When comparing concentrations by sample, bead-based immunoassays generally yielded higher antibody concentrations than the ELISA and had higher variability for serotypes 6B, 18C, and 23F. None of the three assays met the current WHO recommendation of 75% of sera falling within 40% of the assigned antibody concentrations for all seven serotypes. When compared by serotype, the CDC and HPA tests were equivalent for five of seven serotypes, whereas the Luminex assay was equivalent for four of seven serotypes. When overall mean IgG concentrations were compared by laboratory, a higher level of agreement (CCC close to 1) was found among bead-based immunoassays than between the assays and WHO assignments. When compared to WHO assignments, the HPA assay outperformed the other assays (r = 0.920; CCC = 0.894; coefficient of accuracy = 0.972). Additional testing with sera from immunogenicity studies should demonstrate the applicability of this methodology for vaccine evaluation.
doi:10.1128/CVI.00022-10
PMCID: PMC2863388  PMID: 20335434
7.  Kinetics of Lethal Factor and Poly-d-Glutamic Acid Antigenemia during Inhalation Anthrax in Rhesus Macaques ▿  
Infection and Immunity  2009;77(8):3432-3441.
Systemic anthrax manifests as toxemia, rapidly disseminating septicemia, immune collapse, and death. Virulence factors include the anti-phagocytic γ-linked poly-d-glutamic acid (PGA) capsule and two binary toxins, complexes of protective antigen (PA) with lethal factor (LF) and edema factor. We report the characterization of LF, PA, and PGA levels during the course of inhalation anthrax in five rhesus macaques. We describe bacteremia, blood differentials, and detection of the PA gene (pagA) by PCR analysis of the blood as confirmation of infection. For four of five animals tested, LF exhibited a triphasic kinetic profile. LF levels (mean ± standard error [SE] between animals) were low at 24 h postchallenge (0.03 ± 1.82 ng/ml), increased at 48 h to 39.53 ± 0.12 ng/ml (phase 1), declined at 72 h to 13.31 ± 0.24 ng/ml (phase 2), and increased at 96 h (82.78 ± 2.01 ng/ml) and 120 h (185.12 ± 5.68 ng/ml; phase 3). The fifth animal had an extended phase 2. PGA levels were triphasic; they were nondetectable at 24 h, increased at 48 h (2,037 ± 2 ng/ml), declined at 72 h (14 ± 0.2 ng/ml), and then increased at 96 h (3,401 ± 8 ng/ml) and 120 h (6,004 ± 187 ng/ml). Bacteremia was also triphasic: positive at 48 h, negative at 72 h, and positive at euthanasia. Blood neutrophils increased from preexposure (34.4% ± 0.13%) to 48 h (75.6% ± 0.08%) and declined at 72 h (62.4% ± 0.05%). The 72-h declines may establish a “go/no go” turning point in infection, after which systemic bacteremia ensues and the host's condition deteriorates. This study emphasizes the value of LF detection as a tool for early diagnosis of inhalation anthrax before the onset of fulminant systemic infection.
doi:10.1128/IAI.00346-09
PMCID: PMC2715684  PMID: 19506008
8.  Evaluation of Amphotericin B Interpretive Breakpoints for Candida Bloodstream Isolates by Correlation with Therapeutic Outcome 
One hundred seven Candida bloodstream isolates (51 C. albicans, 24 C. glabrata, 13 C. parapsilosis, 13 C. tropicalis, 2 C. dubliniensis, 2 C. krusei, and 2 C. lusitaniae strains) from patients treated with amphotericin B alone underwent in vitro susceptibility testing against amphotericin B using five different methods. Fifty-four isolates were from patients who failed treatment, defined as death 7 to 14 days after the incident candidemia episode, having persistent fever of ≥5 days' duration after the date of the incident candidemia, or the recurrence of fever after two consecutive afebrile days while on antifungal treatment. MICs were determined by using the Clinical Laboratory Standards Institute (formally National Committee for Clinical Laboratory Standards) broth microdilution procedure with two media and by using Etest. Minimum fungicidal concentrations (MFCs) were also measured in two media. Broth microdilution tests with RPMI 1640 medium generated a restricted range of MICs (0.125 to 1 μg/ml); the corresponding MFC values ranged from 0.5 to 4 μg/ml. Broth microdilution tests with antibiotic medium 3 produced a broader distribution of MIC and MFC results (0.015 to 0.25 μg/ml and 0.06 to 2 μg/ml, respectively). Etest produced the widest distribution of MICs (0.094 to 2 μg/ml). However, none of the test formats studied generated results that significantly correlated with therapeutic success or failure.
doi:10.1128/AAC.50.4.1287-1292.2006
PMCID: PMC1426914  PMID: 16569842
9.  Effects of Long-Term Storage on Plasmid Stability in Bacillus anthracis 
Applied and Environmental Microbiology  2005;71(12):7778-7780.
The plasmid profiles of 619 cultures of Bacillus anthracis which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (P < 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (P = 0.25), but there was a statistical interdependence between the two plasmids (P = 0.004). Loss of viability of B. anthracis cultures stored on agar slants is also discussed.
doi:10.1128/AEM.71.12.7778-7780.2005
PMCID: PMC1317469  PMID: 16332750
10.  SARS Surveillance during Emergency Public Health Response, United States, March–July 2003 
Emerging Infectious Diseases  2004;10(2):185-194.
In response to the emergence of severe acute respiratory syndrome (SARS), the United States established national surveillance using a sensitive case definition incorporating clinical, epidemiologic, and laboratory criteria. Of 1,460 unexplained respiratory illnesses reported by state and local health departments to the Centers for Disease Control and Prevention from March 17 to July 30, 2003, a total of 398 (27%) met clinical and epidemiologic SARS case criteria. Of these, 72 (18%) were probable cases with radiographic evidence of pneumonia. Eight (2%) were laboratory-confirmed SARS-coronavirus (SARS-CoV) infections, 206 (52%) were SARS-CoV negative, and 184 (46%) had undetermined SARS-CoV status because of missing convalescent-phase serum specimens. Thirty-one percent (124/398) of case-patients were hospitalized; none died. Travel was the most common epidemiologic link (329/398, 83%), and mainland China was the affected area most commonly visited. One case of possible household transmission was reported, and no laboratory-confirmed infections occurred among healthcare workers. Successes and limitations of this emergency surveillance can guide preparations for future outbreaks of SARS or respiratory diseases of unknown etiology.
doi:10.3201/eid1002.030752
PMCID: PMC3322912  PMID: 15030681
severe acute respiratory syndrome; United States; surveillance; incidence; SARS virus; Coronaviridae; pneumonia; travel; respiratory tract infections
11.  Multilaboratory Evaluation of a Viability Assay for Measurement of Opsonophagocytic Antibodies Specific to the Capsular Polysaccharides of Streptococcus pneumoniae 
Opsonophagocytosis is a correlate of protection that measures the functional activity of vaccine-induced antibodies. A standardized opsonophagocytosis assay (OPA) should be used as part of the evaluation of current and future pneumococcal (Pnc) polysaccharide (Ps)-based vaccines. We enrolled five laboratories to evaluate a previously standardized viability OPA. Each laboratory was provided with a detailed OPA protocol, seven target Pnc strains (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F), two quality control sera and 12 paired sera (blinded) from adult donors who received one dose of the 23-valent Pnc Ps vaccine. Laboratories sent their results to the Centers for Disease Control and Prevention for analysis. Sera were tested in duplicate (single run), and the results were averaged to yield a single OPA titer (≥50% killing) for each serum sample. The percentage of sera within one or two dilutions of the calculated median OPA titer was determined for each laboratory and for each serotype. In general, laboratories were capable of detecting OPA titers within one or two dilutions of the median for at least 75 and 88%, respectively, of the sera tested. The level of agreement with the median OPA titers varied depending on the participating laboratory (overall agreement = 0.8 [99% confidence interval = 0.75 to 0.85]). All OPA median titers reported for quality control sera were within one dilution of the expected titer. We conclude that this OPA can be done in multiple laboratories with a high degree of interlaboratory reproducibility.
doi:10.1128/CDLI.10.6.1019-1024.2003
PMCID: PMC262452  PMID: 14607861
13.  Evaluation of Four Commercially Available Rapid Serologic Tests for Diagnosis of Leptospirosis 
Journal of Clinical Microbiology  2003;41(2):803-809.
Four rapid tests for the serologic diagnosis of leptospirosis were evaluated, and the performance of each was compared with that of the current standard, the microscopic agglutination test (MAT). The four rapid tests were a microplate immunoglobulin M (IgM)-enzyme-linked immunosorbent assay (ELISA), an indirect hemagglutination assay (IHA), an IgM dipstick assay (LDS), and an IgM dot-ELISA dipstick test (DST). A panel of 276 sera from 133 cases of leptospirosis from four different geographic locations was tested as well as 642 sera from normal individuals or individuals with other infectious or autoimmune diseases. Acute-phase sera from cases (n = 148) were collected ≤14 days (median = 6.0) after the onset of symptoms, and convalescent-phase sera (n = 128) were collected ≥15 days after onset (median = 29.1). By a traditional method (two-by-two contingency table), the sensitivities for detection of leptospirosis cases were 93.2% by LDS, 92.5% by DST, 86.5% by ELISA, and 79.0% by IHA. Specificity was 98.8% by DST, 97% by ELISA and MAT, 95.8% by IHA, and 89.6% by LDS. With a latent class analysis (LCA) model that included all the rapid tests and the clinical case definition, sensitivity was 95.5% by DST, 94.5% by LDS, 89.9% by ELISA, and 81.1% by IHA. The sensitivity and specificity estimated by the traditional methods were quite close to the LCA estimates. However, LCA allowed estimation of the sensitivity of the MAT (98.2%), which traditional methods do not allow. For acute-phase sera, sensitivity was 52.7% by LDS, 50.0% by DST, 48.7% by MAT and ELISA, and 38.5% by IHA. The sensitivity for convalescent-phase sera was 93.8% by MAT, 84.4% by DST, 83.6% by LDS, 75.0% by ELISA, and 67.2% by IHA. A good overall correlation with the MAT was obtained for each of the assays, with the highest concordance being with the DST (kappa value, 0.85; 95% confidence interval [CI], 0.8 to 0.90). The best correlation was between ELISA and DST (kappa value, 0.86; 95% CI, 0.81 to 0.91). False-positive LDS results were frequent (≥20%) in sera from individuals with Epstein-Barr virus, human immunodeficiency virus, and periodontal disease and from healthy volunteers. The ease of use and significantly high sensitivity and specificity of DST and ELISA make these good choices for diagnostic testing.
doi:10.1128/JCM.41.2.803-809.2003
PMCID: PMC149700  PMID: 12574287
14.  Specific, Sensitive, and Quantitative Enzyme-Linked Immunosorbent Assay for Human Immunoglobulin G Antibodies to Anthrax Toxin Protective Antigen 
Emerging Infectious Diseases  2002;8(10):1103-1110.
The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 µg/mL, a reliable lower limit of detection of 0.09 µg/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 µg/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 94.2%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.
doi:10.3201/eid0810.020380
PMCID: PMC2730307  PMID: 12396924
Bacillus anthracis; anthrax; antibody; assay; toxin; bioterrorism; ELISA; serology
15.  Reproducibility of Bordetella pertussis Genomic DNA Fragments Generated by XbaI Restriction and Resolved by Pulsed-Field Gel Electrophoresis 
Journal of Clinical Microbiology  2002;40(3):811-816.
The intra- and interlaboratory variabilities of the molecular size measurements of each DNA fragment contributing to three pulsed-field gel electrophoresis (PFGE) profiles were assessed, as were the reproducibilities of the entire PFGE profiles for three Bordetella pertussis strains. The major source of variability within a laboratory occurred between subcultures rather than within gels or between gels. Each PFGE profile was generated reproducibly and was objectively defined by the molecular sizes of its composite fragments. A strain or profile most suitable for use as an internal reference standard was identified.
doi:10.1128/JCM.40.3.811-816.2002
PMCID: PMC120225  PMID: 11880398
16.  Random Amplified Polymorphic DNA Assay as a Rapid Tool in Screening for Neisseria meningitidis Serogroup C Isolates of Electrophoretic Type 24 
Journal of Clinical Microbiology  2001;39(4):1622-1625.
Neisseria meningitidis serogroup C (NMSC) isolates of electrophoretic type 24 (ET-24), as identified by multilocus enzyme electrophoresis, are the main cause of serogroup C meningococcal disease outbreaks and sporadic meningococcal disease in the United States. We evaluated a random amplified polymorphic DNA assay as a rapid tool to screen for isolates of ET-24 by testing 199 NMSC isolates of 51 different ETs. A sensitivity of 88% and a specificity of 87% was achieved in identification of ET-24 isolates when the patterns obtained by two primers, P1 and P5, were analyzed together.
doi:10.1128/JCM.39.4.1622-1625.2001
PMCID: PMC87984  PMID: 11283101
17.  Evaluation of Pulsed-Field Gel Electrophoresis in Epidemiological Investigations of Meningococcal Disease Outbreaks Caused by Neisseria meningitidis Serogroup C 
Since 1990, the frequency of Neisseria meningitidis serogroup C (NMSC) outbreaks in the United States has increased. Based on multilocus enzyme electrophoresis (MEE), the current molecular subtyping standard, most of the NMSC outbreaks have been caused by isolates of several closely related electrophoretic types (ETs) within the ET-37 complex. We chose 66 isolates from four well-described NMSC outbreaks that occurred in the United States from 1993 to 1995 to evaluate the potential of pulsed-field gel electrophoresis (PFGE) to identify outbreak-related isolates specific for each of the four outbreaks and to differentiate between them and 50 sporadic isolates collected during the outbreak investigations or through active laboratory-based surveillance from 1989 to 1996. We tested all isolates collected during the outbreak investigations by four other molecular subtyping methods: MEE, ribotyping (ClaI), random amplified polymorphic DNA assay (two primers), and serotyping and serosubtyping. Among the 116 isolates, we observed 11 clusters of 39 NheI PFGE patterns. Excellent correlation between the PFGE and the epidemiological data was observed, with an overall sensitivity of 85% and specificity of 71% at the 95% pattern relatedness breakpoint using either 1.5 or 1.0% tolerance. For all four analyzed outbreaks, PFGE would have given public health officials additional support in declaring an outbreak and making appropriate public health decisions.
doi:10.1128/JCM.39.1.75-85.2001
PMCID: PMC87683  PMID: 11136752
18.  An Analytical Model Applied to a Multicenter Pneumococcal Enzyme-Linked Immunosorbent Assay Study 
Journal of Clinical Microbiology  2000;38(6):2043-2050.
Pneumococcal conjugate vaccines will eventually be licensed after favorable results from phase III efficacy trials. After licensure of a conjugate vaccine for invasive pneumococcal disease in infants, new conjugate vaccines will likely be licensed primarily on the basis of immunogenicity data rather than clinical efficacy. Analytical methods must therefore be developed, evaluated, and validated to compare immunogenicity results accurately within and between laboratories for different vaccines. At present no analytical technique is uniformly accepted and used in vaccine evaluation studies to determine the acceptable level of agreement between a laboratory result and the assigned value for a given serum sample. This multicenter study describes the magnitude of agreement among 12 laboratories quantifying an identical series of 48 pneumococcal serum specimens from 24 individuals (quality-control sera) by a consensus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) developed for this study. After provisional or trial antibody concentrations were assigned to the quality-control serum samples for this study, four methods for comparison of a series of laboratory-determined values with the assigned concentrations were evaluated. The percent error between assigned values and laboratory-determined concentrations proved to be the most informative of the four methods. We present guidelines that a laboratory may follow to analyze a series of quality-control sera to determine if it can reproduce the assigned antibody concentrations within an acceptable level of tolerance. While this study focused on a pneumococcal IgG ELISA, the methods that we describe are easily generalizable to other immunological assays.
PMCID: PMC86724  PMID: 10834951
19.  A Modified Enzyme-Linked Immunosorbent Assay for Measurement of Antibody Responses to Meningococcal C Polysaccharide That Correlate with Bactericidal Responses 
The standardized enzyme-linked immunosorbent assay (ELISA) for measurement of serum immunoglobulin G (IgG) antibody responses to meningococcal C polysaccharide has been modified to employ assay conditions that ensure specificity and favor detection primarily of high-avidity antibodies. The modified and standard assays were used to measure IgG antibody concentrations in sera of toddlers vaccinated with meningococcal polysaccharide vaccine or a meningococcal C conjugate vaccine. The results were compared to the respective complement-mediated bactericidal antibody titers. In sera obtained after one or two doses of vaccine, the correlation coefficients, r, for the results of the standard assay and bactericidal antibody titers were 0.45 and 0.29, compared to 0.85 and 0.87, respectively, for the modified assay. With the standard assay, there were no significant differences between the geometric mean antibody responses of the two vaccine groups. In contrast, with the modified assay, 5- to 20-fold higher postvaccination antibody concentrations were measured in the conjugate than in the polysaccharide group. Importantly, the results of the modified assay, but not the standard ELISA, paralleled the respective geometric mean bactericidal antibody titers. Thus, by employing conditions that favor detection of higher-avidity IgG antibody, the modified ELISA provides results that correlate closely with measurements of antibody functional activity that are thought to be important in protection against meningococcal disease.
PMCID: PMC95603  PMID: 9665952
20.  Age-Dependent Neisseria meningitidis Serogroup C Class-Specific Antibody Concentrations and Bactericidal Titers in Sera from Young Children from Montana Immunized with a Licensed Polysaccharide Vaccine 
Infection and Immunity  1998;66(6):2453-2459.
Neisseria meningitidis serogroup C bactericidal titers and class-specific enzyme-linked immunosorbent assay (ELISA) antibody concentrations were measured in sera from 173 children (1 to 5 years old) before and 6 weeks and 7 months following vaccination with a quadrivalent (A/C/Y/W-135) polysaccharide vaccine. The immune responses of the children were compared with those of 40 adults 6 weeks postvaccination. Both bactericidal titers and ELISA antibody concentrations were significantly higher in the adults than in the children (P < 0.05). In addition, the ratio of immunoglobulin G (IgG) to IgM was higher in the children than in the adults. With an ELISA total antibody concentration of ≥2 μg/ml used as a measure of seroconversion, ≥84% of the individuals from each age group responded to the serogroup C polysaccharide. However, with a ≥4-fold-increase in bactericidal titer used, only 18% of 1-year-olds, 32% of 2-year-olds, and 50 to 60% of 3-, 4-, and 5-year-olds seroconverted. The ELISA results suggest that >50% of all children retained ≥2 μg of total antibody per ml at 7 months postimmunization. However, the bactericidal titers suggest that <10% of children <4 years old retained a ≥4-fold increase at 7 months following vaccination. Of particular note, 59 of 79 sera (75%) from the 1- and 2-year-olds had high ELISA antibody concentrations (2 to 20 μg/ml) with no associated bactericidal titer (<1:8). Discordant results between bactericidal titers and ELISA antibody concentrations were not explained by the presence of IgA blocking antibody or relative levels of IgG and IgM. The bactericidal results show age-dependent differences in the production and retention of antibody in young children immunized with serogroup C polysaccharide; these differences are not evident with the ELISA data.
PMCID: PMC108224  PMID: 9596702

Results 1-20 (20)