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Serum bactericidal activity (SBA) and ELISA antibody levels elicited by two efficacious serogroup B meningococcal vaccines were measured in a controlled trial involving 408 15- to 20-year-olds. Subjects were given two doses at a 6-week interval of a serogroup B or control vaccine. Response was defined as > or = 4-fold rise in antibody level. After two doses of the Finlay Institute (Havana) vaccine at 12 months, the proportions of SBA and ELISA responders were not different from those of the control group (15% and 17% [vaccine] vs. 13% and 9% [control], P > .05). After two doses of the National Institute of Public Health (Oslo) vaccine, there were more SBA and ELISA responders than in the control group (47% and 34% [vaccine] vs. 10% and 1% [control]) or the Finlay Institute vaccine group (P < .05 for both). SBA and ELISA may be insensitive correlates for protective efficacy for some outer membrane protein-based serogroup B meningococcal vaccines. [ABSTRACT FROM AUTHOR]

Vaccine immunogenicity and reactogenicity depend on recipient and vaccine characteristics. We hypothesized that healthy adults reporting higher reactogenicity from seasonal inactivated influenza vaccine (IIV) developed higher antibody titers compared with those reporting lower reactogenicity. We performed a secondary analysis of a randomized phase 1 trial of a trivalent IIV delivered by microneedle patch (MNP) or intramuscular (IM) injection. We created composite reactogenicity scores as exposure variables and used hemagglutination inhibition (HAI) titers as outcome variables. We used mixed-model analysis of variance to estimate geometric mean titers (GMTs) and titer fold change and modified Poisson generalized estimating equations to estimate risk ratios of seroprotection and seroconversion. Estimates of H3N2 GMTs were associated with the Systemic and Local scores among the IM group. Within the IM group, those with high reaction scores had lower baseline H3N2 GMTs and twice the titer fold change by day 28. Those with high Local scores had a greater probability of seroconversion. These results suggest that heightened reactogenicity to IM IIV is related to low baseline humoral immunity to an included antigen. Participants with greater reactogenicity developed greater titer fold change after 4 weeks, although the response magnitude was similar or lower compared with low-reactogenicity participants.

Background: Pertussis serodiagnosis is increasingly being used in the United States despite the lack of a US Food and Drug Administration-approved, commercially available assay. To better understand the utility of these assays in diagnosing pertussis, serology assays were evaluated for analytical parameters and clinical accuracy., Methods: Forty-three antigen-antibody combinations were evaluated for single-point diagnosis of pertussis. Serum panels included sera from laboratory-confirmed cases, an international reference standard, and healthy donors. Phase I panel (n = 20) of sera was used to assess precision, linearity, and accuracy; Phase II panel (n = 226) followed with positive percent agreement (PPA) and negative percent agreement (NPA) estimates. Analytical analyses included coefficients of variation (CV) and concordance correlation coefficients (rc)., Results: Intra-analyst variability was found to be relatively low among samples per assay, with only 6% (78 of 1240) having CV >20%, primarily with the highly concentrated immunoglobulin (Ig)G anti-pertussis toxin (PT) specimens and IgM assays. The rc measurements to assess linearity ranged between 0.282 and 0.994, 0.332 and 0.999, and -0.056 and 0.482 for IgA, IgG, and IgM, respectively. Analytical accuracy for calibrated IgG anti-PT assays was 86%-115%. The PPA and NPA varied greatly for all assays; PPA/NPA ranges for IgA, IgG, and IgM assays, with culture and/or polymerase chain reaction positivity as control, were 29-90/13-100, 26-96/27-100, and 0-73/42-100, respectively. In IgG assays, mixing filamentous hemagglutinin antigen with PT increased PPA but decreased NPA., Conclusions: Seroassays varied substantially under both analytical and clinical parameters; however, those that were calibrated to a reference standard were highly accurate. Our findings support incorporation of calibrated pertussis seroassays to the pertussis case definition for improved diagnosis and surveillance., (Published by Oxford University Press on behalf of the Pediatric Infectious Diseases Society 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Opsonophagocytic assays (OPAs) are routinely used for assessing the immunogenicity of pneumococcal vaccines, with OPA data often being utilized for licensure of new vaccine formulations. However, no reference serum for pneumococcal OPAs is available, making evaluation of data among different laboratories difficult. This international collaboration was initiated to (i) assign consensus opsonic indexes (OIs) to FDA pneumococcal reference serum lot 007sp (here referred to as 007sp) and a panel of serum samples used for calibration of the OPA and (ii) determine if the normalization of the OPA results obtained with test samples to those obtained with 007sp decreases the variability in OPA results among laboratories. To meet these goals, six participating laboratories tested a panel of serum samples in five runs for 13 serotypes. For each serum sample, consensus OIs were obtained using a mixed-effects analysis of variance model. For the calibration serum samples, normalized consensus values were also determined on the basis of the results obtained with 007sp. For each serotype, the overall reduction in interlaboratory variability was calculated by comparing the coefficients of variation of the unadjusted and the normalized values. Normalization of the results substantially reduced the interlaboratory variability, ranging from a 15% reduction in variability for serotype 9V to a 64% reduction for serotype 7F. Normalization also increased the proportion of data within 2-fold of the consensus value from approximately 70% (average for all serotypes) to >90%. On the basis of the data obtained in this study, pneumococcal reference standard lot 007sp will likely be a useful reagent for the normalization of pneumococcal OPA results from different laboratories. The data also support the use of the 16 FDA serum samples used for calibration of the OPA as part of the initial evaluation of new assays or periodic assessment of established assays., (Copyright © 2017 American Society for Microbiology.)

Protective antigen (PA)-specific antibody and cell-mediated immune (CMI) responses to annual and alternate booster schedules of anthrax vaccine adsorbed (AVA; BioThrax) were characterized in humans over 43 months. Study participants received 1 of 6 vaccination schedules: a 3-dose intramuscular (IM) priming series (0, 1, and 6 months) with a single booster at 42 months (4-IM); 3-dose IM priming with boosters at 18 and 42 months (5-IM); 3-dose IM priming with boosters at 12, 18, 30, and 42 months (7-IM); the 1970 licensed priming series of 6 doses (0, 0.5, 1, 6, 12, and 18 months) and two annual boosters (30 and 42 months) administered either subcutaneously (SQ) (8-SQ) or IM (8-IM); or saline placebo control at all eight time points. Antibody response profiles included serum anti-PA IgG levels, subclass distributions, avidity, and lethal toxin neutralization activity (TNA). CMI profiles included frequencies of gamma interferon (IFN-γ)- and interleukin 4 (IL-4)-secreting cells and memory B cells (MBCs), lymphocyte stimulation indices (SI), and induction of IFN-γ, IL-2, IL-4, IL-6, IL-1β, and tumor necrosis factor alpha (TNF-α) mRNA. All active schedules elicited high-avidity PA-specific IgG, TNA, MBCs, and T cell responses with a mixed Th1-Th2 profile and Th2 dominance. Anti-PA IgG and TNA were highly correlated (e.g., month 7,r(2)= 0.86,P< 0.0001, log10 transformed) and declined in the absence of boosters. Boosters administered IM generated the highest antibody responses. Increasing time intervals between boosters generated antibody responses that were faster than and superior to those obtained with the final month 42 vaccination. CMI responses to the 3-dose IM priming remained elevated up to 43 months. (This study has been registered at ClinicalTrials.gov under registration no. NCT00119067.)., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Integrated Management of Childhood Illness (IMCI) guidelines were developed to decrease morbidity and mortality, yet implementation varies across settings. Factors associated with poor adherence are not well understood., Methods: We used data from Manhiça District Hospital outpatient department and five peripheral health centers to examine pneumonia management for children <5 years old from January 2008 to June 2011. Episodes of IMCI-defined pneumonia (cough or difficult breathing plus tachypnea), severe pneumonia (pneumonia plus chest wall in-drawing), and/or clinician-diagnosed pneumonia (based on discharge diagnosis) were included., Results: Among severe pneumonia episodes, 96.2% (2,918/3,032) attended in the outpatient department and 70.0% (291/416) attended in health centers were appropriately referred to the emergency department. Age<1 year, malnutrition and various physical exam findings were associated with referral. For non-severe pneumonia episodes, antibiotics were prescribed in 45.7% (16,094/35,224). Factors associated with antibiotic prescription included age <1 year, abnormal auscultatory findings, and clinical diagnosis of pneumonia; diagnosis of malaria or gastroenteritis and pallor were negatively associated with antibiotic prescription., Conclusion: Adherence to recommended management of severe pneumonia was high in a hospital outpatient department, but suboptimal in health centers. Antibiotics were prescribed in fewer than half of non-severe pneumonia episodes, and diagnosis of malaria was the strongest risk factor for incorrect management., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Humoral and cell-mediated immune correlates of protection (COP) for inhalation anthrax in a rhesus macaque (Macaca mulatta) model were determined. The immunological and survival data were from 114 vaccinated and 23 control animals exposed to Bacillus anthracis spores at 12, 30, or 52 months after the first vaccination. The vaccinated animals received a 3-dose intramuscular priming series (3-i.m.) of anthrax vaccine adsorbed (AVA) (BioThrax) at 0, 1, and 6 months. The immune responses were modulated by administering a range of vaccine dilutions. Together with the vaccine dilution dose and interval between the first vaccination and challenge, each of 80 immune response variables to anthrax toxin protective antigen (PA) at every available study time point was analyzed as a potential COP by logistic regression penalized by least absolute shrinkage and selection operator (LASSO) or elastic net. The anti-PA IgG level at the last available time point before challenge (last) and lymphocyte stimulation index (SI) at months 2 and 6 were identified consistently as a COP. Anti-PA IgG levels and lethal toxin neutralization activity (TNA) at months 6 and 7 (peak) and the frequency of gamma interferon (IFN-γ)-secreting cells at month 6 also had statistically significant positive correlations with survival. The ratio of interleukin 4 (IL-4) mRNA to IFN-γ mRNA at month 6 also had a statistically significant negative correlation with survival. TNA had lower accuracy as a COP than did anti-PA IgG response. Following the 3-i.m. priming with AVA, the anti-PA IgG responses at the time of exposure or at month 7 were practicable and accurate metrics for correlating vaccine-induced immunity with protection against inhalation anthrax., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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.

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 r(2) = 0.89 for log(10)-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.

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.

Lot 89SF has been the reference standard serum pool used in pneumococcal enzyme-linked immunosorbent assays (ELISAs) since 1990. In 2005, it was estimated that there remained between 2 and 5 years' supply of lot 89SF. Since lot 89SF was the reference standard used in the evaluation of the seven-valent pneumococcal conjugate vaccine Prevnar (PCV7), the link to clinical efficacy would be severed if stocks became completely depleted. Furthermore, demonstration of immune responses comparable to those elicited by PCV7 is a licensure approach used for new pneumococcal conjugate vaccines, so a replacement reference standard was required. A total of 278 volunteers were immunized with the 23-valent unconjugated polysaccharide vaccine Pneumovax II, and a unit of blood was obtained twice within 120 days following immunization. Plasma was prepared, pooled, and confirmed to be free from hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV. The pooled serum was poured at 6 ml per vial into 15,333 vials and lyophilized. Immunological bridging of 007sp to 89SF was used to establish equivalent reference values for 13 pneumococcal capsular serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) by five independent laboratories. Antibody concentrations in 007sp were established relative to the lot 89SF reference preparation using the WHO reference ELISA. Subsequently, 12 existing WHO calibration sera had concentrations reassigned for 13 pneumococcal serotypes using new serum 007sp as the reference, and these were compared to concentrations relative to the original reference serum. Agreement was excellent for the 12 WHO calibration sera. The 007sp preparation has replaced 89SF as the pneumococcal reference standard. Sufficient quantity of this new preparation is available such that, with judicious use, it should be available for at least 25 years.

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.

Objectives: We examined associations between the socioeconomic characteristics of census tracts and racial/ethnic disparities in the incidence of bacteremic community-acquired pneumonia among US adults., Methods: We analyzed data on 4870 adults aged 18 years or older with community-acquired bacteremic pneumonia identified through active, population-based surveillance in 9 states and geocoded to census tract of residence. We used data from the 2000 US Census to calculate incidence by age, race/ethnicity, and census tract characteristics and Poisson regression to estimate rate ratios (RRs) and 95% confidence intervals (CIs)., Results: During 2003 to 2004, the average annual incidence of bacteremic pneumonia was 24.2 episodes per 100 000 Black adults versus 10.1 per 100 000 White adults (RR = 2.40; 95% CI = 2.24, 2.57). Incidence among Black residents of census tracts with 20% or more of persons in poverty (most impoverished) was 4.4 times the incidence among White residents of census tracts with less than 5% of persons in poverty (least impoverished). Racial disparities in incidence were reduced but remained significant in models that controlled for age, census tract poverty level, and state., Conclusions: Adults living in impoverished census tracts are at increased risk of bacteremic pneumonia and should be targeted for prevention efforts.

Systemic anthrax manifests as toxemia, rapidly disseminating septicemia, immune collapse, and death. Virulence factors include the anti-phagocytic gamma-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.

A double-blind, randomized, controlled phase I study to assess the safety, immunogenicity, and antibody persistence of a new group A conjugate vaccine (PsA-TT) in volunteers aged 18 to 35 years was previously performed. Subjects received one dose of either the PsA-TT conjugate vaccine, meningococcal A/C polysaccharide vaccine (PsA/C), or tetanus toxoid vaccine. The conjugate vaccine was shown to be safe and immunogenic as demonstrated by a standardized group A-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and by a serum bactericidal antibody (SBA) assay using rabbit complement (rSBA). This report details further analysis of the sera using four additional immunologic assays to investigate the relationship between the different immunoassays. The immunoassays used were an SBA assay that used human complement (hSBA), a group A-specific IgG multiplexed bead assay, and two opsonophagocytic antibody (OPA) assays which used two different methodologies. For each vaccine group, geometric mean concentrations or geometric mean titers were determined for all assays before and 4, 24, and 48 weeks after vaccination. Pearson's correlation coefficients were used to assess the relationship between the six assays using data from all available visits. An excellent correlation was observed between the group A-specific IgG concentrations obtained by ELISA and those obtained by the multiplexed bead assay. hSBA and rSBA titers correlated moderately, although proportions of subjects with putatively protective titers and those demonstrating a > or = 4-fold rise were similar. The two OPA methods correlated weakly and achieved only a low correlation with the other immunoassays. The correlation between hSBA and group A-specific IgG was higher for the PsA-TT group than for the PsA/C group.

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 >or=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 microg/ml); the corresponding MFC values ranged from 0.5 to 4 microg/ml. Broth microdilution tests with antibiotic medium 3 produced a broader distribution of MIC and MFC results (0.015 to 0.25 microg/ml and 0.06 to 2 microg/ml, respectively). Etest produced the widest distribution of MICs (0.094 to 2 microg/ml). However, none of the test formats studied generated results that significantly correlated with therapeutic success or failure.

Background: The African meningitis belt undergoes recurrent epidemics caused by Neisseria meningitidis serogroup A. During 2002, Burkina Faso documented the first large serogroup W-135 (NmW-135) meningococcal disease epidemic. To understand the emergence of NmW-135, we investigated meningococcal carriage and immunity., Methods: Immediately after Burkina Faso's epidemic, we conducted a cross-sectional survey of meningococcal carriage and seroprevalence in an epidemic and a nonepidemic district. We identified predictors of elevated NmW-135 serum bactericidal activity (SBA), a functional correlate of protection, using multivariate logistic regression., Results: The NmW-135 carriage rate was 25.2% in the epidemic district and 3.4% in the nonepidemic district (P<.0001). Compared with residents of the nonepidemic district, those of the epidemic district had higher geometric mean titers of NmW-135 SBA (P<.0001). NmW-135 SBA titers>or=1:8, an estimated protective threshold, were observed in 60.4% and 34.0% of residents of the epidemic and nonepidemic district, respectively (P=.0002). In a multivariate model, current NmW-135 carriage, age, and residence in the epidemic district were independent predictors of having an NmW-135 SBA titer>or=1:8., Conclusions: Extensive NmW-135 carriage and transmission in the epidemic area caused residents to acquire natural immunity. Serial carriage and seroprevalence surveys could establish the duration of immunity in the population. The persistent circulation of NmW-135 underscores the potential for periodic NmW-135 epidemics in Africa.

Objective: To determine the mortality, hospital stay, and total hospital charges and cost of hospitalization attributable to candidemia by comparing patients with candidemia with control-patients who have otherwise similar illnesses. Prior studies lack broad patient and hospital representation or cost-related information that accurately reflects current medical practices., Design: Our case-control study included case-patients with candidemia and their cost-related data, ascertained from laboratory-based candidemia surveillance conducted among all residents of Connecticut and Baltimore and Baltimore County, Maryland, during 1998 to 2000. Control-patients were matched on age, hospital type, admission year, discharge diagnoses, and duration of hospitalization prior to candidemia onset., Results: We identified 214 and 529 sets of matched case-patients and control-patients from the two locations, respectively. Mortality attributable to candidemia ranged between 19% and 24%. On multivariable analysis, candidemia was associated with mortality (OR, 5.3 for Connecticut and 8.5 for Baltimore and Baltimore County; P < .05), whereas receiving adequate treatment was protective (OR, 0.5 and 0.4 for the two locations, respectively; P < .05). Candidemia itself did not increase the total hospital charges and cost of hospitalization; when treatment status was accounted for, having received adequate treatment for candidemia significantly increased the total hospital charges and cost of hospitalization ($6,000 to $29,000 and $3,000 to $22,000, respectively) and the length of stay (3 to 13 days)., Conclusion: Our findings underscore the burden of candidemia, particularly regarding the risk of death, length of hospitalization, and cost associated with treatment.

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.

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 (> or = 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.

Persons with acquired immunodeficiency syndrome (AIDS) have a higher incidence of invasive pneumococcal disease (IPD) than other adults, and many receive long-term trimethoprim-sulfamethoxazole (TMP-SMZ) prophylactic therapy. We used 1998-1999 data from the Active Bacterial Core surveillance of the Emerging Infections Program Network to compare IPD infections between adults aged 18-64 years with human immunodeficiency virus (HIV) infection and other adults. Of 2346 patients with IPD, 416 (18%) had HIV or AIDS (HIV/AIDS). Certain serotypes (serotypes 6A, 6B, 9N, 9V, 18C, 19A, 19F, and 23F) were more common among patients with HIV/AIDS than in adults with no underlying disease (P<.05, vs. serotype 4), even when TMP-SMZ-nonsusceptible isolates were excluded. HIV/AIDS (adjusted odds ratio [aOR], 1.93; 95% confidence interval [CI], 1.44-2.59), immunocompromising conditions other than HIV/AIDS (aOR, 1.56; 95% CI, 1.12-2.18), and black race (aOR, 1.50; 95% CI, 1.20-1.88) were independent risk factors for infection with these serotypes. HIV/AIDS was not an independent risk factor for TMP-SMZ nonsusceptibility. Vulnerability to certain serotypes among adults with HIV/AIDS may have implications in prevention strategies.

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 or=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 (>or=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.

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 micro g/mL, a reliable lower limit of detection of 0.09 micro g/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 micro g/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 97.6%. 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.

Objective: To assess the sensitivity, specificity and predictive value positive of the WHO threshold strategy for detecting meningococcal disease epidemics in sub-Saharan Africa and to estimate the impact of the strategy on an epidemic at district level., Methods: Data on meningitis cases at the district level were collected weekly from health ministries, WHO country and regional offices, and nongovernmental organizations in countries where there were epidemics of meningococcal disease in 1997. An epidemic was defined as a cumulative district attack rate of at least 100 cases per 100,000 population from January to May, the period of epidemic risk. The sensitivity, specificity and predictive value positive of the WHO threshold rate were calculated, and curves of sensitivity against (1 - specificity) were compared with alternatively defined threshold rates and epidemic sizes. The impact of the WHO strategy on a district epidemic was estimated by comparing the numbers of epidemic cases with cases estimated to have been prevented by vaccination., Findings: An analysis was made of 48 198 cases reported in 174 districts in Benin, Burkina Faso, the Gambia, Ghana, Mali, Niger, and Togo. These cases were 80.3% of those reported from Africa to WHO during the 1997 epidemic period. District populations ranged from 10,298 to 573,908. The threshold rate was crossed during two consecutive weeks in 69 districts (39.7%) and there were epidemics in 66 districts (37.9%). Overall, the sensitivity of the threshold rate for predicting epidemics was 97%, the specificity was 95%, and the predictive value positive was 93%. Taken together, these values were equivalent or better than the sensitivity, specificity and predictive value positive of alternatively defined threshold rates and epidemics, and remained high regardless of district size. The estimated number of potential epidemic cases decreased by nearly 60% in the age group targeted for vaccination in one district where the guidelines were followed in a timely manner., Conclusion: The use of the WHO strategy was sensitive and specific for the early detection of meningococcal disease epidemics in countries of sub-Saharan Africa during 1997 and had a substantial impact on a district epidemic. Nevertheless, the burden of meningococcal disease in these countries remains formidable and additional control measures are needed.

To determine national trends in mortality due to invasive mycoses, we analyzed National Center for Health Statistics multiple-cause-of-death record tapes for the years 1980 through 1997, with use of their specific codes in the International Classification of Diseases, Ninth Revision (ICD-9 codes 112.4-118 and 136.3). In the United States, of deaths in which an infectious disease was the underlying cause, those due to mycoses increased from the tenth most common in 1980 to the seventh most common in 1997. From 1980 through 1997, the annual number of deaths in which an invasive mycosis was listed on the death certificate (multiple-cause [MC] mortality) increased from 1557 to 6534. In addition, rates of MC mortality for the different mycoses varied markedly according to human immunodeficiency virus (HIV) status but were consistently higher among males, blacks, and persons > or =65 years of age. These data highlight the public health importance of mycotic diseases and emphasize the need for continuing surveillance.

Surveillance for coccidioidomycosis (CM) and a case-control study for risk factors among adults were conducted in Kern County, California. From January 1995 through December 1996, 905 cases of CM were identified, for an annual incidence of 86 cases per 100,000 population. A total of 380 adults were enrolled in the case-control study: 77 had severe pulmonary disease, 33 had disseminated disease, and 270 control patients had mild disease. Independent risk factors for severe pulmonary disease included diabetes, recent history of cigarette smoking, income of < $15,000 per year, and older age. Oral antifungal therapy before hospitalization was associated with a reduced risk of CM pneumonia. Risk factors for disseminated disease were black race, income of < $15,000 per year, and pregnancy. Early treatment of CM with oral antifungal agents may prevent severe pulmonary disease in groups considered to be at high risk, such as elderly individuals, persons with diabetes, and smokers. Persons at risk for severe CM may benefit from vaccination once an effective CM vaccine is available.

Two hundred eighty-one sporadic Neisseria meningitidis serogroup B isolates, collected through active laboratory-based surveillance, were selected to be analyzed by PorA variable region (VR) typing to determine the prevalence of PorA types in the United States. A substantial number of distinct VR types were identified, 31 in VR1 and 41 in VR2. A total of 73 different PorA types were found, and 76. 7% of these types comprise nonprototype sequences in VR1, VR2, or both. The most prevalent PorA types were P1.7,16-20 (previously P1.7, 16i), P1.22,14, P1.22-1,14 (previously P1.22a,14), P1.7,16, P1.7-1,1 (previously P1.7d,1), P1.19,15, and P1.17,16-3 (previously P1.B,16d). No correlation was observed between the PorA types and geographic origin of the isolates. These data may aid in the design of an efficacious outer membrane protein-based vaccine by identifying the most appropriate PorA types for vaccine formulation. Studies are needed to fully evaluate the extent of cross-protection in humans among the variants and prototypes in each PorA VR family.

Leptospirosis is a zoonotic disease of worldwide distribution caused by spirochetes of the genus Leptospira. Humans are infected through direct contact with infected animals or through exposure to fresh water or soil contaminated by infected animal urine. Leptospirosis is characterized by acute fever that can be followed by a more severe, sometimes fatal illness that may include jaundice and renal failure (Weil's disease), meningitis, myocarditis, hemorrhagic pneumonitis, or hemodynamic collapse. To identify potential risk factors for leptospirosis in Thailand, we conducted a matched case-control study in Nakornratchasrima Province of the northeastern region. Fifty-nine cases and 118 controls were included in the study. Four activities in the two weeks prior to illness were independently associated with leptospirosis infection: walking through water (odds ratio [OR] = 4.9, 95% confidence interval [CI] = 1.7-14.1), applying fertilizer in wet fields for more than 6 hr a day (OR = 3.4, 95% CI = 1.5-7.8), plowing in wet fields for more than 6 hr a day (OR = 3.5, 95% CI = 1.1-11.6), and pulling out rice plant sprouts in wet fields for more than 6 hr a day (OR = 3.1, 95% CI = 1.02-9.3). Identification of these risk factors on admission might prove useful for early diagnosis and treatment of leptospirosis in Thailand.

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.

Because of the increase in incidence of coccidioidomycosis among the elderly in Arizona between 1990 and 1996, a case-control study was conducted to look at risk factors for disease among these persons. Cases (n=89) were persons aged > or =60 years with laboratory-confirmed coccidioidomycosis; 2 control groups were selected, the first by use of random-digit dialing (geographic controls, n=91) and the second by use of lists of persons with negative serologic coccidioidomycosis tests (laboratory-negative controls, n=58). Elderly persons with coccidioidomycosis had spent significantly less time in Arizona than did persons in either control group and were more likely than geographic controls to have congestive heart failure or cancer, to have smoked, or to have taken corticosteroids. Elderly persons who recently have moved to Arizona or who have chronic illnesses and their physicians need to be aware of their higher risk for coccidioidomycosis in order to improve their chances of early diagnosis and treatment. These persons may benefit from vaccination, once an effective vaccine for coccidioidomycosis is developed.

From 1 January 1995 through 31 June 1997, 153 cases of coccidioidomycosis in human immunodeficiency virus (HIV)-infected persons were identified in Arizona (incidence, 41/1000 persons living with AIDS). A case-control study was conducted to evaluate risk factors for coccidioidomycosis in HIV-infected persons. A case was defined as laboratory-confirmed, incident coccidioidomycosis in a person infected with HIV for > or =3 months, and each case patient had 3 control patients matched by county, age group, sex, HIV/AIDS status, and CD4 lymphocyte count. Multivariable analysis identified black race and a history of oropharyngeal or esophageal candidiasis to be associated with increased risk of coccidioidomycosis; protease inhibitor therapy was associated with a reduced risk. In persons with previous history of oropharyngeal or esophageal candidiasis, having received an azole drug was associated with a reduced risk (odds ratio, 0.4; 95% confidence interval, 0.2-0.9; P=.04). Physicians may need to consider azole chemoprophylaxis for HIV-infected persons who live in areas of endemicity, have CD4 cell counts <200/microL, are black, or have a history of thrush.

The pneumococcal polysaccharide vaccine is recommended as a means of preventing invasive disease in the elderly. We compared responses to the 23-valent polysaccharide vaccine in 46 previously unvaccinated, healthy, institutionalized elderly persons (mean age, 85.5 years) with those in 12 healthy younger adults (mean age, 37 years) by measuring prevaccination and postvaccination serum IgG antibody concentrations (by ELISA), functional antibody activity (by opsonophagocytosis), IgG antibody avidity, and passive protection in mice. Postvaccination IgG antibody concentrations for two serotypes (6B and 19F) of the five studied (4, 6B, 14, 19F, and 23F) were significantly lower in elderly than in younger adults; however, opsonophagocytic activity was significantly reduced for all serotypes in the elderly. Sera with reduced opsonophagocytic activity (titer, <64) correlated with low IgG antibody avidity and protected mice poorly against pneumococcal challenge. In elderly persons receiving polysaccharide vaccination, there was a significant reduction in the functionality of postvaccination antibodies, and this appeared to increase with advanced age.

Objectives: The purpose of this study was to evaluate the effectiveness of Haemophilus influenzae type b (Hib) conjugate vaccines among children aged 2 to 18 months and to determine risk factors for invasive Hib disease during a period of declining incidence (1991-1994)., Methods: A prospective population-based case-control study was conducted in a multistate US population of 15.5 million. A laboratory-based active surveillance system was used for case detection., Results: In a multivariate analysis, having a single-parent mother (odds ratio [OR] = 4.3, 95% confidence interval [CI] = 1.2, 14.8) and household crowding (OR = 3.5, 95% CI = 1.03, 11.7) were risk factors for Hib disease independent of vaccination status. After adjustment for these risk factors, the protective efficacy of 2 or more Hib vaccine doses was 86% (95% CI = 16%, 98%). Among undervaccinated subjects, living with a smoker (P = .02) and several indicators of lower socioeconomic status were risk factors for Hib disease., Conclusions: Hib disease still occurs at low levels in the United States, predominantly in socioeconomically disadvantaged populations. Low immunization coverage may facilitate continuing transmission of Hib. Special efforts to achieve complete and timely immunization in disadvantaged populations are needed.

Despite near elimination of Haemophilus influenzae b (Hib) meningitis from several industrialized countries following introduction of conjugate Hib vaccines into infant immunization schedules, Hib remains a major cause of meningitis and pneumonia in resource-poor countries. In Niger, Hib causes nearly 200 cases of meningitis per 100,000 children < one year of age, and > 40% of cases are fatal. We evaluated the immunogenicity of Hib polysaccharide-tetanus toxoid conjugate vaccine (PRP-T) administered in the same syringe as diphtheria-tetanus-pertussis (DTP) vaccine among infants in Niger. Infants were randomized into group 1 (PRP-T at six, 10, and 14 weeks), group 2 (PRP-T at 10 and 14 weeks), or a control group (meningococcal A/C polysaccharide vaccine). By 14 weeks of age, all subjects in groups land 2 had > or = 0.15 microg/ml of anti-PRP antibody, and 82% versus 76% had > or = 1.0 microg/ml of antibody (P=not significant). By nine months of age the proportion of infants with > or = 0.15 and > or = 1.0 microg/ml was group I=97% and 76%; group 2=93% and 67%; controls=10% and 2.6%. Four weeks after the first, second, and third doses of PRP-T, infants in group 1 showed geometric mean titers (GMTs) of 0.19, 3.97, and 6.09 microg/ml while infants in group 2 had GMTs of 2.40 and 4.41 microg/ml four weeks after the delayed first and second doses. Both PRP-T groups had significantly higher GMTs at 18 weeks and nine months of age than infants in the control group. The Hib PRP-T vaccine was immunogenic in infants in Niger. The strong response after PRP-T was initiated one month after the first DTP vaccination may reflect carrier priming. Two dose schedules of PRP-T should be given serious consideration, particularly if their reduced cost permits vaccine introduction that would be otherwise unaffordable.

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.

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 microg/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 microg 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 microg/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.

Clinical characteristics associated with bacillary angiomatosis and bacillary peliosis (BAP) in patients with human immunodeficiency virus (HIV) infection were evaluated in a case-control study; 42 case-patients and 84 controls were matched by clinical care institution. Case-patients presented with fever (temperature, > 37.8 degrees C; 93%), a median CD4 lymphocyte count of 21/mm3, cutaneous or subcutaneous vascular lesions (55%), lymphadenopathy (21%), and/or abdominal symptoms (24%). Many case-patients experienced long delays between medical evaluation and diagnosis of BAP (median, 4 weeks; range, 1 day to 24 months). Case-patients were more likely than controls to have fever, lymphadenopathy, hepatomegaly, splenomegaly, a low CD4 lymphocyte count, anemia, or an elevated serum level of alkaline phosphatase (AP) (P < .001). In multivariate analysis, a CD4 lymphocyte count of < 200/mm3 (matched odds ratio [OR], 9.9; P < .09), anemia reflected by a hematocrit value of < 0.36 (OR, 19.7; P < .04), and an elevated AP level of > or = 2.6 mukat/L (OR, 23.9; P < .05) remained associated with disease after therapy with zidovudine was controlled for. BAP should be considered an AIDS-defining opportunistic infection and should be included in the differential diagnosis for febrile, HIV-infected patients with cutaneous or osteolytic lesions, lymphadenopathy, abdominal symptoms, anemia, or an elevated serum level of AP.

A new standard meningococcal reference serum designated CDC1992 was prepared to replace meningococcal reference sera ECG and PB-2, which are not available in sufficient quantities for continued use as primary reference sera. CDC1992 was prepared from 14 healthy adult volunteers who underwent plasmapheresis 4 to 12 weeks postvaccination with a single dose of a Neisseria meningitidis quadrivalent polysaccharide vaccine. Total and/or class-specific meningococcal serogroup A and C anticapsular antibody concentrations (in micrograms per milliliter) were assigned to CDC1992 by using homologous and heterologous enzyme-linked immunosorbent assay (ELISA) formats. The reference serum ECG was used as a reference standard to assign total anticapsular antibody concentrations to CDC1992 by a homologous ELISA format. A heterologous ELISA format, with the Haemophilus influenzae type b standard reference serum FDA 1983, was used to assign total and class-specific antibody concentrations to CDC1992. Alkaline phosphatase-labeled mouse anti-human monoclonal antibody conjugates were used as secondary antibodies in both ELISA formats. The total, immunoglobulin G (IgG), IgA, and IgM antibody concentrations, assigned to CDC1992 for serogroup A were 135.8, 91.8, 20.1, and 23.9 micrograms/ml, respectively, and those for serogroup C were 32.0, 24.1, 5.9, and 2.0 micrograms/ml, respectively. Meningococcal serogroup A and C antibody concentrations were in good agreement when homologous and heterologous ELISA format results were compared. Total and class-specific serogroup A and C antibody concentrations were determined in six adult quality control serum samples from the Centers for Disease Control and Prevention by using the homologous ELISA and our assigned antibody concentrations for CDC1992. Antibody concentrations in reference sera ECG and PB-2 were measured in order to provide a historical link to previous studies. The general acceptance of CDC1992 as the standard reference serum and the assigned antibody concentrations will allow investigators to compare antibody levels in serum to those in a single reference preparation.

Ribotyping was compared with multilocus enzyme electrophoresis (MEE) for subtyping 305 Listeria monocytogenes isolates from clinical and nonclinical sources. For ribotyping, EcoRI-restricted genomic DNA fragments of L. monocytogenes strains were separated by agarose gel electrophoresis, and Southern blots were probed with a cloned Escherichia coli rrnB operon (plasmid pKK3535) labeled with digoxigenin. The L. monocytogenes isolates were divided into 28 distinct ribotypes, while MEE analysis divided the same isolates into 78 electrophoretic types (ETs). On the basis of their ribotype profiles, the strains were divided into two subgroups. The ribotype alpha (RT alpha) subgroup contained serotypes 1/2a, 1/2c, and 3a, and the ribotype beta (RT beta) subgroup contained serotypes 1/2b, 3b, 4b, and 4ab. This division is in complete agreement with MEE analysis, which divides the species into two subgroups (ET groups A and B), with the same serotype distribution in each subgroup. Overall, MEE was more discriminating than ribotyping. However, in several instances ribotyping discriminated between isolates within the same ET. Ribotyping was more discriminating for serotypes 1/2a, 1/2c, and 3a (Simpson's Index for Diversity [DI] = 0.81) than for serotypes 1/2b and 4b (DI = 0.76). A substantial proportion (69%) of serotype 1/2b and 4b strains clustered in five ETs and five ribotypes. These data suggest that ribotyping and MEE do not provide adequate discrimination between strains of serotypes 1/2b and 4b. Methods such as pulsed-field gel electrophoresis and random amplified polymorphic DNA analysis should be explored for further discrimination of strains of these serotypes.

There is a lack of consensus among investigators who use a variety of immunoassay techniques (e.g., enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay) regarding the protocols for describing and forming standard reference or calibration curves and interpolating serum antibody concentrations. This confounds the issue of detecting the presence or absence of parallelism between standard reference serum and serially diluted serum sample curves. These curves must be parallel to support the assumption that the antibody-binding characteristics are similar enough to allow the determination of antibody levels in the diluted serum sample. There is no universal and widely adopted strategy for assessing parallelism in bioassays, and without an assurance of parallelism, investigators are not able to calculate reliable estimates for antibody concentrations in serum samples. Furthermore, single-point (dilution) serum assays do not provide information related to parallelism and nonparallelism, and this, too, may lead to considerable error when calculating antibody concentrations. When assay methodology, technique, and precision improve to the extent that standard reference serum and serially diluted serum sample curves are fit with little error, standard analysis of variance techniques are overly sensitive to negligible departures from parallelism. We present a series of guidelines that compose a protocol for assessing parallelism between bioassay dilution curves that are applicable to data derived from ELISAs. These criteria should be applicable, with minor modifications, to most immunoassay experimental situations and, most importantly, are not dependent on the mathematical model used to form the standard reference curve. These guidelines have evolved in our laboratories over the past 4 years during the performance of thousands of ELISAs for antibodies to the capsular polysaccharides of Neisseria meningitidis groups A and C and Haemophilus influenzae type b.

Listeria bacteremia occurred in 2 pregnant women whose only common exposure was attendance at a party. The incubation period, the possibility of mild disease due to Listeria infection, and foods associated with risk of disease were evaluated. Ten (28%) of 36 party attenders met a case definition, which included isolation of Listeria monocytogenes from blood or stool or two of the following: fever, musculoskeletal symptoms, nausea, vomiting, diarrhea. One of 25 stool cultures was positive. The 2 blood isolates and 1 stool isolate were serotype 4b and identical by enzyme typing. The incubation periods for illness in the 2 pregnant women were 19 and 23 days. Consumption of large amounts of shrimp, nonalcoholic beverages, Camembert cheese, and cauliflower was significantly associated with illness. Eating shrimp remained a significant risk factor for illness after controlling for consumption of other foods. This study suggests a milder illness may exist in healthy persons who consume foods contaminated with L. monocytogenes and demonstrates a prolonged incubation period for disease.

A standardized enzyme-linked immunosorbent assay (ELISA) was used by 11 laboratories to measure levels of total serum antibody to Neisseria meningitidis serogroup C capsular polysaccharide in 16 unpaired pre- and postvaccination serum samples. Twelve serum samples were from adults, and four were from children aged 2, 3, 5, and 9. The between-laboratory coefficient of variation for pre- and postvaccination sera ranged from 16 to 59% and 11 to 21%, respectively. The average percent difference (absolute value) from the between-laboratory means for all prevaccination sera measured by each laboratory was 24%, whereas the average percent difference was 13% for all postvaccination sera. A postvaccination quality control serum was diluted three times to give optical densities on the high, middle, and low portions of the standard reference curve. The three dilutions were assayed by the 11 laboratories a total of 241 times and yielded an overall coefficient of variation of 20%. Antibody-binding inhibition curves showed that the standardized ELISA was specific for N. meningitidis serogroup C capsular polysaccharide antibody. Fifty percent inhibition of seven serum samples was obtained after reaction with an average concentration of 0.9 micrograms of meningococcal serogroup C polysaccharide per ml; an average of 93% inhibition was obtained with 50 micrograms of polysaccharide per ml. The acceptance and use of this standardized ELISA will reduce between-laboratory assay variability and ensure a more accurate and reproducible assessment of immunogenicity for vaccines under development.

The long-term kinetics of the immunologic response after vaccination of adults with Neisseria meningitidis polysaccharide vaccine is unknown. Total meningococcal anti-capsular antibody response (measured by ELISA) and serum bactericidal activity after routine vaccination with quadrivalent meningococcal vaccine were evaluated in US Air Force personnel. In a retrospective cross-sectional study, blood samples were obtained from approximately 40 personnel before vaccination, at 1 and 4-6 months, and at 2, 3, 4, 6, 8, and 10 years after vaccination. Total anti-group A and -group C capsular antibody levels and bactericidal activity peaked 1 month after vaccination and declined substantially by 2 years. At each interval, significantly higher levels of total antibody and bactericidal activity were detected than before vaccination. Anti-capsular antibodies and bactericidal activity persisted for up to 10 years after immunization. These and further studies on the serologic measure of protection against meningococcal disease are important for evaluation of candidate vaccines and development of recommendations for immunization.

To estimate the prevalence of Neisseria meningitidis relatively resistant to penicillin in the United States, antimicrobial susceptibility testing was performed on all US meningococcal isolates submitted to the Centers for Disease Control and Prevention in 1991, including isolates identified through population-based surveillance for invasive meningococcal disease in selected areas of the United States. Three of the 100 isolates tested had MICs of penicillin of 0.125 microgram/mL. All were serogroup B, beta-lactamase-negative, and unique by multilocus enzyme electrophoresis subtyping. None of the 3 patients had been treated solely with penicillin; all recovered completely. About 4% of the isolates obtained from the population-based surveillance system were relatively penicillin-resistant. Given the low prevalence and uncertain clinical significance of infection with these organisms, routine susceptibility testing of meningococcal isolates is not indicated at this time; however, continued surveillance is necessary to monitor trends in antimicrobial susceptibility of meningococci in the United States.

Antibodies to group A meningococcal polysaccharide were measured by hemagglutination (HA) and by ELISA in sera obtained from Gambian children before vaccination and 3 weeks, 2 years, and 5 years after vaccination with a group A + group C meningococcal capsular polysaccharide vaccine. Children were 1-4 years old at the time of vaccination. Most showed a good initial response to vaccination, including those aged 1-2 years. However, antibody titers declined progressively during follow-up, and 5 years after vaccination, antibody titers measured by both HA and ELISA had returned to prevaccination levels. This decline was not influenced significantly by a booster dose of vaccine given 2 years after initial immunization. Administration of malaria chemoprophylaxis reduced the rate at which antibody levels fell after initial immunization. Sustained protection of children against group A meningococcal disease will require the development of vaccines that are immunogenic in infants and that can induce T cell memory.

Three selective enrichment procedures-the U.S. Food and Drug Administration (FDA) method, the U.S. Department of Agriculture (USDA) method, and the Netherlands Government Food Inspection Service (NGFIS) method-were compared for isolating Listeria monocytogenes from contaminated foods. The foods were obtained from the refrigerators of patients with culture-proven listeriosis who were identified through multistate active surveillance in a U.S. population of 19 million. The study was designed to identify foods that may be important in transmission of L. monocytogenes in sporadic cases of human listeriosis. Of 899 foods analyzed by all three methods, 121 were positive for L. monocytogenes by at least one method. The three enrichment methods detected L. monocytogenes in 65% (FDA), 74% (USDA), and 74% (NGFIS) of the foods shown to contain L. monocytogenes . The differences among the three methods were not statistically significant. However, the recovery of L. monocytogenes by a combination of any two methods (USDA-FDA 88%, USDA-NGFIS 91%, FDA-NGFIS 87%) was significantly better than that by one method alone (p < 0.02). The differences among the combinations of methods were not statistically significant. These results suggest that at least two enrichment methods must be used in combination to recover L. monocytogenes from contaminated foods with a success rate near 90%. Correlations were observed between negative results and low (<0.3 CFU/g) level of L. monocytogenes contamination for the USDA (p << 0.001) and NGFIS (p << 0.001) methods. A similar but somewhat weaker association was observed for the FDA method (p < 0.06).

We present a computerized pattern recognition model used to speciate mycobacteria based on their restriction fragment length polymorphism (RFLP) banding patterns. DNA fragment migration distances were normalized to minimize lane-to-lane variability of band location both within and among gels through the inclusion of two internal size standards in each sample. The computer model used a library of normalized RFLP patterns derived from samples of known origin to create a probability matrix which was then used to classify the RFLP patterns from samples of unknown origin. The probability matrix contained the proportion of bands that fell within defined migration distance windows for each species in the library of reference samples. These proportions were then used to compute the likelihood that the banding pattern of an unknown sample corresponded to that of each species represented in the probability matrix. As a test of this process, we developed an automated, computer-assisted model for the identification of Mycobacterium species based on their normalized RFLP banding patterns. The probability matrix contained values for the M. tuberculosis complex, M. avium, M. intracellulare, M. kansasii and M. gordonae species. Thirty-nine independent strains of known origin, not included in the probability matrix, were used to test the accuracy of the method in classifying unknowns: 37 of 39 (94.9%) were classified correctly. An additional set of 16 strains of known origin representing species not included in the model were tested to gauge the robustness of the probability matrix. Every sample was correctly identified as an outlier, i.e. a member of a species not included in the original matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

A two-step assay combining a gene amplification step and a restriction fragment length polymorphism analysis was developed to differentiate the Mycobacterium species that account for greater than 90% of potentially pathogenic isolates and greater than 86% of all isolates in clinical laboratories in the United States. These species are M. tuberculosis, M. bovis, M. avium, M. intracellulare, M. kansasii, and M. gordonae. With lysates of pure cultures as the template, two oligonucleotide primers that amplified an approximately 1,380-bp portion of the hsp65 gene from all 139 strains of 19 Mycobacterium species tested, but not from the 19 non-Mycobacterium species tested, were identified. Digestion of the amplicons from 126 strains of the six most commonly isolated Mycobacterium species with the restriction enzymes BstNI and XhoI in separate reactions generated restriction fragment patterns that were distinctive for each of these species, except for those of M. tuberculosis and M. bovis, which were not distinguishable. By including size standards in each sample, the restriction fragment profiles could be normalized to a fixed distance and the similarities of patterns could be calculated by using a computer-aided comparison program. The availability of this data base should enable the identification of an unknown Mycobacterium strain to the species level by a comparison of the restriction fragment pattern of the unknown with the data base of known patterns.

There is no standard immunoassay for evaluating immune responses to meningococcal vaccines. We developed an enzyme-linked immunosorbent assay to measure total levels of antibody to Neisseria meningitidis group A capsular polysaccharide. Five laboratories measured the antibody levels in six paired pre- and postvaccination serum samples by using the enzyme-linked immunosorbent assay. Methylated human serum albumin was used to bind native group A polysaccharide to microtiter plate surfaces. The between-laboratory coefficients of variation for pre- and postvaccination sera had ranges of 31 to 91 and 17 to 31, respectively. The mean laboratory coefficients of variation for pre- and postvaccination sera, respectively, were 17 and 11 (Molecular Biology Laboratory, Centers for Disease Control), 12 and 15 (Immunodiagnostic Methods Laboratory, Centers for Disease Control), 22 and 19 (Dana-Farber Cancer Institute), 38 and 38 (Bacterial Polysaccharide Laboratory, U.S. Food and Drug Administration), and 11 and 10 (Praxis Biologics, Inc.). Standardization of this enzyme-linked immunosorbent assay should allow interlaboratory comparison of meningococcal vaccine immunogenicity, thus providing a laboratory-based assessment tool for evaluating meningococcal vaccines.

Whole-cell chromosomal digests of 84 strains of aerotolerant Campylobacter (AC) were examined by using PvuII restriction fragment length polymorphisms of rRNA genes followed by hybridization with Escherichia coli 16S and 23S rRNA (ribotyping). The AC strains belonged to Campylobacter cryaerophila (n = 13) and a newly defined species, "C. butzleri" (n = 64). Strains of C. cryaerophila belonged to two hybridization groups: DNA group 1A (including the type strain of C. cryaerophila) and DNA group 1B (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). Six AC strains not classified as C. cryaerophila or "C. butzleri" were also included. All 35 sporadic human and animal isolates of "C. butzleri" sent to the Centers for Disease Control for identification showed different ribotype patterns. However, most "C. butzleri" strains contained common bands at approximately 3.0, 6.2, 12.0, and 15.0 kb; the 3.0-kb band was present in all but four strains. An additional 23 strains of "C. butzleri," isolated as part of special studies, contained the 3.0-kb band. Thus, on the basis of visual identification of the 3.0-kb band, 94% of available strains were correctly identified as "C. butzleri." Ribotyping demonstrated that C. cryaerophila strains (DNA groups 1A and 1B) were different from C. butzleri strains. All C. cryaerophila strains demonstrated a common ribosomal DNA restriction fragment of 3.2 kb; DNA group 1B strains contained an additional common band at 2.6 kb. Ribotyping patterns of AC species were easily distinguished from patterns of other Campylobacter, Helicobacter, and Wolinella species.(ABSTRACT TRUNCATED AT 250 WORDS)