Freyja Lynn

Use of Coefficient of Variation in Assessing Variability of Quantitative Assays

ABSTRACT We have derived the mathematical relationship between the coefficient of variation associated with repeated measurements from quantitative assays and the expected fraction of pairs of those measurements that differ by at least some given factor, i.e., the expected frequency of disparate results that are due to assay variability rather than true differences. Knowledge of this frequency helps determine what magnitudes of differences can be expected by chance alone when the particular coefficient of variation is in effect. This frequency is an operational index of variability in the sense that it indicates the probability of observing a particular disparity between two measurements under the assumption that they measure the same quantity. Thus the frequency or probability becomes the basis for assessing if an assay is sufficiently precise. This assessment also provides a standard for determining if two assay results for the same subject, separated by an intervention such as vaccination or infection, differ by more than expected from the variation of the assay, thus indicating an intervention effect. Data from an international collaborative study are used to illustrate the application of this proposed interpretation of the coefficient of variation, and they also provide support for the assumptions used in the mathematical derivation.

Establishment of Diagnostic Cutoff Points for Levels of Serum Antibodies to Pertussis Toxin, Filamentous Hemagglutinin, and Fimbriae in Adolescents and Adults in the United States

ABSTRACT Numerous reports have documented that serologic methods are much more sensitive than culture for the diagnosis of pertussis in adolescents and adults. However, a standardized serologic test for pertussis is not routinely available to most clinicians, and the serologic test levels or cutoff points correlated with diseases have not been determined. The goal of the present study was to examine the distribution of immunoglobulin G (IgG) levels against three Bordetella pertussis antigens (pertussis toxin [PT], filamentous hemagglutinin [FHA], and fimbria types 2 and 3 [FIM]) and to determine population-based antibody levels for the purpose of establishing such diagnostic cutoff points. Enzyme-linked immunosorbent assays (ELISAs) were performed with sera from >6,000 U.S. residents aged 6 to 49 years who participated in the Third National Health and Nutrition Examination Survey. Mixture models were developed to identify hypothesized exposure groups and establish diagnostic cutoffs. Quantifiable (>20 ELISA units/ml [EU]) anti-FHA and anti-FIM IgG antibodies were common (65 and 62% of individuals, respectively), but quantifiable anti-PT IgG antibodies were less frequent (16%). Given the distributions of antibody levels, an anti-PT IgG level of ≥94 EU was proposed as the diagnostic cutoff point. Application of this cutoff point to culture-confirmed illness in a prior study investigating cough illness yielded a high diagnostic sensitivity (80%) and specificity (93%). A standardized ELISA for anti-PT IgG with a single serum sample appears to be useful for the identification of recent B. pertussis infection in adolescents and adults with cough illness. The PT cutoff point will be further evaluated in prospective studies of confirmed B. pertussis infection.

Effect of O Acetylation of Neisseria meningitidis Serogroup A Capsular Polysaccharide on Development of Functional Immune Responses

ABSTRACT The importance of O-acetyl groups to the immunogenicity of Neisseria meningitidis serogroup A polysaccharide (PS) was examined in studies using human sera and mouse immunization. In 17 of 18 postimmunization human sera, inhibition enzyme-linked immunosorbent assay indicated that the majority of antibodies binding to serogroup A PS were specific for epitopes involving O-acetyl groups. Studies with mice also showed an essential role for O-acetyl groups, where serum bactericidal titers following immunization with de-O-acetylated (de-O-Ac) conjugate vaccine were at least 32-fold lower than those following immunization with O-Ac PS-conjugate vaccine and 4-fold lower than those following immunization with native capsular PS. Inhibition studies using native and de-O-Ac PS confirmed the specificity of murine antibodies to native PS. The dramatic reduction in immunogenicity associated with removal of O-acetyl groups indicates that O acetylation is essential to the immunogenic epitopes of serogroup A PS. Since levels of bactericidal antibodies are correlated with protection against disease, O-acetyl groups appear to be important in protection.

Interlaboratory Comparison of Results of an Anthrax Lethal Toxin Neutralization Assay for Assessment of Functional Antibodies in Multiple Species

ABSTRACT The anthrax lethal toxin neutralization assay (TNA) will likely be used to correlate the protection offered by new anthrax vaccines in animal models to the immunogenicity that will be provided in humans. TNA data are being generated in several different laboratories to measure the immune responses in rabbits, nonhuman primates, and humans. In order to compare data among species and laboratories, a collaborative study was conducted in which 108 samples from the three species were analyzed in seven independent laboratories. Six of the seven laboratories had participated in an interlaboratory technology transfer of the TNA. Analysis of the titration curves generated by samples from each species indicated that the behaviors of the samples from all species were similar; the upper and lower asymptotes and the slopes of the curves were less than 30% divergent from those for human reference material. Dilutional linearity was consistent among samples from each species, with spike to effective dilution at 50% inhibition (ED50) slopes of less than 1.2 for all species. Agreement among the laboratories with consensus values was within 10% of the ED50s for all samples and within 7.5% of the quotients of the test sample ED50 and the reference standard ED50 (NF50s) for all samples. The relative standard deviations obtained when data from all laboratories and for all species were combined were 45% for the ED50s and 35% for the NF50s. These precision data suggest that the NF50 readout may normalize the values generated by different laboratories. This study demonstrates that the TNA is a panspecies assay that can be performed in several different laboratories with a high degree of quantitative agreement and precision.

Genetic Typing of the Porin Protein of Neisseria gonorrhoeae from Clinical Noncultured Samples for Strain Characterization and Identification of Mixed Gonococcal Infections

ABSTRACT Molecular methods that characterize the Neisseria gonorrhoeae porin protein Por are needed to study gonococcal pathogenesis in the natural host and to classify strains from direct clinical samples used with nucleic acid amplification-based tests. We have defined the capabilities of por variable region (VR) typing and determined suitable conditions to apply the method to direct clinical specimens. Nested PCR from spiked urine samples detected 1 to 10 copies of template DNA; freezing spiked whole urine greatly reduced the ability to amplify porB. In a laboratory model of mixed gonococcal infections, the por type of one strain could be determined in the presence of a 100-fold excess of another. por VR typing was used to examine clinical samples from women enrolled in studies conducted in Baltimore, Md., and Madagascar. por type was determined from 100% of paired cervical swab and wick samples from 20 culture-positive women from Baltimore; results for eight individuals (40%) suggested infection with more than one strain. In frozen urine samples from Madagascar, porB was amplified and typed from 60 of 126 samples from ligase chain reaction (LCR)-positive women and 3 samples from LCR-negative women. The por VR types of 13 samples (21%) suggested the presence of more than one gonococcal strain. Five por types, identified in >45% of women with typed samples, were common to both geographic areas. Molecular typing is an important adjunct to nucleic acid amplification-based diagnostics. Methods that utilize direct clinical samples and can identify mixed infections may contribute significantly to studies of host immunity, gonococcal epidemiology, and pathogenesis.

Utilization of Serologic Assays to Support Efficacy of Vaccines in Nonclinical and Clinical Trials: Meeting at the Crossroads

In May 2009 the National Institute of Allergy and Infectious Diseases hosted a workshop on serologic assays that support vaccine efficacy evaluations. The meeting promoted exchange of ideas among investigators from varying disciplines who are working on anti-infectious agent vaccines at different stages of development. The presentations and discussions at the workshop illustrated the challenges common across various pathogens with recurring themes: (1) A thorough understanding of the science regarding the pathogen and the host response to disease and immunization is fundamental to assay selection. (2) The intended use of the immunoassay data must be clearly defined to ensure appropriate specificity, accuracy, and precision; a laboratory must also commit resources to assure data quality and reliability. (3) During vaccine development, an immunoassay may evolve with respect to quality, purpose, and degree of standardization, and, in some cases, must be changed or replaced as data are accumulated. (4) Collaboration on standardized reagents and methods, harmonization efforts, and multidisciplinary teams facilitate consistent generation of quality data. This report provides guidance for effective development and utilization of immunoassays based on the lessons learned from currently licensed vaccines. Investigators are encouraged to create additional opportunities for scientific exchange, noting that the discussed themes are relevant for immunoassays used for other purposes such as therapeutics and diagnostics.

por variable-region typing by DNA probe hybridization is broadly applicable to epidemiologic studies of Neisseria gonorrhoeae

ABSTRACT The porin gene (porB) of Neisseria gonorrhoeae encodes the major outer membrane protein identified as PI or Por. To examine the utility of por variable-region (VR) typing, porB from 206 isolates was characterized by using oligonucleotide probes in a checkerboard hybridization assay that identifies the sequence types of five VRs of both PIA and PIB porB alleles. The strains represented temporally and geographically distinct isolates, isolates from a large cluster, epidemiologically linked partner isolates, and a collection of strains from disseminated gonococcal infections. By using rigorous epidemiologic criteria for transmission of infection between sex partners, por VR typing was more discriminatory than serovar typing in classifying isolates from both members of 43 epidemiologically linked pairs: 39 of 43 pairs were classified as coinciding by por VR typing compared to 43 of 43 by serovar determination (P = 0.058). porB sequence data confirmed the accuracy of the por VR method. Relationships between VR type and serovar typing monoclonal antibodies were observed for all six PIB and three of six PIA antibodies. por VR typing is a molecular tool that appears to have broad applicability. This method can be adapted to a wide range of technologies from simple hybridization to microarray and may allow for typing from noncultured clinical specimens.

Development and use of a serum bactericidal assay using pooled human complement to assess responses to a meningococcal group A conjugate vaccine in African toddlers

ABSTRACT A meningococcal group A polysaccharide (PS) conjugate vaccine (PsA-TT) has been developed for African countries affected by epidemic meningitis caused by Neisseria meningitidis. Complement-mediated serum bactericidal antibody (SBA) assays are used to assess protective immune responses to meningococcal vaccination. Human complement (hC′) was used in early studies demonstrating antibody-mediated protection against disease, but it is difficult to obtain and standardize. We developed and evaluated a method for sourcing hC′ and then used the SBA assay with hC′ (hSBA) to measure bactericidal responses to PsA-TT vaccination in 12- to 23-month-old African children. Sera with active complement from 100 unvaccinated blood donors were tested for intrinsic bactericidal activity, SBA titer using rabbit complement (rSBA), and anti-group A PS antibody concentration. Performance criteria and pooling strategies were examined and then verified by comparisons of three independently prepared hC′ lots in two laboratories. hSBA titers of clinical trial sera were then determined using this complement sourcing method. Two different functional antibody tests were necessary for screening hC′. hSBA titers determined using three independent lots of pooled hC′ were within expected assay variation among lots and between laboratories. In African toddlers, PsA-TT elicited higher hSBA titers than meningococcal polysaccharide or Hib vaccines. PsA-TT immunization or PS challenge of PsA-TT-primed subjects resulted in vigorous hSBA memory responses, and titers persisted in boosted groups for over a year. Quantifying SBA using pooled hC′ is feasible and showed that PsA-TT was highly immunogenic in African toddlers.

Neisseria meningitidis Endotoxin and Capsule Transmission by Transplantation

To the Editor: Donor organs for transplantation are in extremely short supply. The increased number of patients with advanced liver disease requiring transplantation has resulted in the expansion of selection criteria for potential donors. Previously unacceptable or marginally acceptable donors, such as those who die from bacterial meningitis, are now donor candidates (1). Several case reports and small, retrospective studies have shown that, with appropriate antimicrobial drug treatment, transplant recipients of organs from donors with bacterial meningitis do not have increased risk for infection (2,3) or reduced graft survival (3). However, endotoxemia in liver transplant recipients has been associated with graft failure and a high mortality rate (4,5), and in gram-negative bacterial infections, the physiologic effects of endotoxin may persist after adequate antimicrobial drug treatment. We report a case of liver transplantation in which the donor had brain death from meningococcal meningitis. This unique case provided an opportunity to study the serologic responses and clinical course of an organ recipient after liver transplantation from a donor who had died of Neisseria meningitidis infection. Our observations suggest that biologically relevant levels of antigens, including endotoxin, may have been transferred to the recipient. A 57-year-old woman with progressive sclerosing cholangitis and cryptogenic cirrhosis received a liver transplant from a previously healthy 18-year-old man who died of serogroup C N. meningitidis meningitis. He had received antimicrobial drug therapy with ceftriaxone and ampicillin for 5 days before brain death was determined; cultures were negative, and the mildly elevated liver function tests, recorded on admission, had resolved, and no evidence of hepatic impairment was shown. The transplantation surgery was prolonged (17 h) and technically difficult, requiring intraoperative blood products (≈25 U), prolonged postoperative mechanical ventilation, blood pressure support, and renal hemofiltration. Pathologic examination of the recipients explanted liver showed secondary biliary cirrhosis. The recipient was given ceftriaxone before and after transplantation for 7 days. During postoperative week 4, she was also treated for Pseudomonas aeruginosa nosocomial pneumonia and pleural effusion caused by Enterococcus. She was extubated 3 weeks after transplantation and discharged 5 weeks after transplantation. No evidence of clinical infection with N. meningitidis was identified. Pathologic examination of the explanted donor liver demonstrated focal acute subcapsular necrosis, large droplet fat accumulation, and mild chronic portal inflammation. The N. meningitidis isolated from the blood and cerebrospinal fluid of the donor was serogroup C by immunoprecipitation, and the lipooligosaccharide (LOS) immunotypes (determined by Brenda Brandt, Walter Reed Army Institute of Research, Washington, DC.) were L2, L3, L7, and L9. Banked serum specimens, obtained from the recipient before the operation and every week for 5 weeks after the operation, were assayed for presence of antibodies to N. meningitidis. Elevated levels of immunoglobulin (Ig) G antibodies (11.2 μg/mL at week 2; 22.5 μg/mL at week 4) (6) to the group C meningococcal polysaccharide capsule were detected in recipient serum (Figure, panel A). Levels of IgM antibodies to LOS L9 rose sharply and peaked in the sample taken 2 weeks after the operation (Figure, panel B). Levels of IgM antibodies to L3 also rose slightly between weeks 2 and 4. IgG antibodies to the 4 LOS types and antibodies to outer membrane vesicles were elevated in the first week postoperation and then declined (data not shown) and may reflect antibodies present from intraoperative blood products. Antipolysaccharide antibodies to Streptococcus pneumoniae serotypes 14 and 23 and N. meningitidis serogroup A also rose between weeks 2 and 4 posttransplantation but were not above values expected in normal adult sera at any time. Bactericidal assays against the infecting strain could not be performed because of endogenous killing that was not complement mediated and was presumed to be caused by the presence of antimicrobial drugs in the serum samples. Figure Pre- and posttransplantation serum antibodies as measured by enzyme-linked immunosorbent assay (ELISA). A) Immunoglobulin (Ig) G antibodies to Neisseria meningitidis serogroup C capsular polysaccharide (CPS) determined as described by Arakere and Frasch ... The rise in IgM antibodies to LOS L9 and IgG antibodies to group C polysaccharide is consistent with a response to exposure to Neisseria antigens at the time of transplantation. With effective antimicrobial drug treatment, the recipient has little risk for bacteremia after transplantation of organs from donors dying of N. meningitidis infection (3). However, bacterial antigens, endotoxin, and cytokines could potentially be sequestered in a donor liver, especially when organ transplantation occurs within days of the bacteremic episode. Despite appropriate antimicrobial drug treatment of the donor and recipient, and the absence of any evidence of active infection of the recipient, these data suggest that proinflammatory endotoxin and capsular polysaccharide from N. meningitidis were transplanted with the donor liver. Although we cannot definitively associate these findings with the organ recipients difficult intra- and postoperative course, this case raises the question of the role of proinflammatory responses to transplanted endotoxin in postoperative condition and graft dysfunction in this critically ill population (9,10). Prospective studies identifying and quantifying endotoxin in the transplanted liver itself and in the recipient may be valuable in assessing the meaning of this finding. An assessment of endotoxin transfer will assist in further defining the risks associated with organ transplantation from donors with N. meningitidis infections and may lead to the consideration of additional interventions to mediate the effects of endotoxin exposure.