Assignment of Opsonic Values to Pneumococcal Reference Serum 007sp for Use in Opsonophagocytic Assays for 13 Serotypes.

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.

Establishment of a new human pneumococcal standard reference serum, 007sp.

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.

Advancing the development of diagnostic tests and biomarkers for tuberculosis.

High costs and limited returns on investment have hampered progress in developing new diagnostic tests and treatments for tuberculosis (TB). We need new biomarkers to develop assays that can rapidly, efficiently and reliably detect Mycobacterium tuberculosis infection and disease, identify drug resistance and expedite drug and vaccine development. This can only be accomplished through cross-disciplinary collaborations to facilitate access to human specimens. The Food and Drug Administration, Centers for Disease Control and Prevention, National Institutes of Health, the industry and academia experts came together in a June 2010 workshop to examine the field of TB diagnostic test development and biomarker discovery, identify areas of most urgent need and formulate strategies to address those needs.

Investigation of different group A immunoassays following one dose of meningococcal group A conjugate vaccine or A/C polysaccharide vaccine in adults.

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.

On the use of zero-inflated and hurdle models for modeling vaccine adverse event count data.

We compared several modeling strategies for vaccine adverse event count data in which the data are characterized by excess zeroes and heteroskedasticity. Count data are routinely modeled using Poisson and Negative Binomial (NB) regression but zero-inflated and hurdle models may be advantageous in this setting. Here we compared the fit of the Poisson, Negative Binomial (NB), zero-inflated Poisson (ZIP), zero-inflated Negative Binomial (ZINB), Poisson Hurdle (PH), and Negative Binomial Hurdle (NBH) models. In general, for public health studies, we may conceptualize zero-inflated models as allowing zeroes to arise from at-risk and not-at-risk populations. In contrast, hurdle models may be conceptualized as having zeroes only from an at-risk population. Our results illustrate, for our data, that the ZINB and NBH models are preferred but these models are indistinguishable with respect to fit. Choosing between the zero-inflated and hurdle modeling framework, assuming Poisson and NB models are inadequate because of excess zeroes, should generally be based on the study design and purpose. If the study's purpose is inference then modeling framework should be considered. For example, if the study design leads to count endpoints with both structural and sample zeroes then generally the zero-inflated modeling framework is more appropriate, while in contrast, if the endpoint of interest, by design, only exhibits sample zeroes (e.g., at-risk participants) then the hurdle model framework is generally preferred. Conversely, if the study's primary purpose it is to develop a prediction model then both the zero-inflated and hurdle modeling frameworks should be adequate.

Multistate evaluation of invasive pneumococcal diseases in adults with human immunodeficiency virus infection: serotype and antimicrobial resistance patterns in the United States.

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.

Early detection and response to meningococcal disease epidemics in sub-Saharan Africa: appraisal of the WHO strategy.

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.

Trends in mortality due to invasive mycotic diseases in the United States, 1980-1997.

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.

Random amplified polymorphic DNA assay as a rapid tool in screening for Neisseria meningitidis serogroup C isolates of electrophoretic type 24.

Neisseria meningitidis serogroup C (NMSC) isolates of electrophoretic type 24 (ET-24), as identified by multilocus enzyme electrophoresis, are the main cause of serogroup C meningococcal disease outbreaks and sporadic meningococcal disease in the United States. We evaluated a random amplified polymorphic DNA assay as a rapid tool to screen for isolates of ET-24 by testing 199 NMSC isolates of 51 different ETs. A sensitivity of 88% and a specificity of 87% was achieved in identification of ET-24 isolates when the patterns obtained by two primers, P1 and P5, were analyzed together.

Risk factors for severe pulmonary and disseminated coccidioidomycosis: Kern County, California, 1995-1996.

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.

Evaluation of pulsed-field gel electrophoresis in epidemiological investigations of meningococcal disease outbreaks caused by Neisseria meningitidis serogroup C.

Since 1990, the frequency of Neisseria meningitidis serogroup C (NMSC) outbreaks in the United States has increased. Based on multilocus enzyme electrophoresis (MEE), the current molecular subtyping standard, most of the NMSC outbreaks have been caused by isolates of several closely related electrophoretic types (ETs) within the ET-37 complex. We chose 66 isolates from four well-described NMSC outbreaks that occurred in the United States from 1993 to 1995 to evaluate the potential of pulsed-field gel electrophoresis (PFGE) to identify outbreak-related isolates specific for each of the four outbreaks and to differentiate between them and 50 sporadic isolates collected during the outbreak investigations or through active laboratory-based surveillance from 1989 to 1996. We tested all isolates collected during the outbreak investigations by four other molecular subtyping methods: MEE, ribotyping (ClaI), random amplified polymorphic DNA assay (two primers), and serotyping and serosubtyping. Among the 116 isolates, we observed 11 clusters of 39 NheI PFGE patterns. Excellent correlation between the PFGE and the epidemiological data was observed, with an overall sensitivity of 85% and specificity of 71% at the 95% pattern relatedness breakpoint using either 1.5 or 1.0% tolerance. For all four analyzed outbreaks, PFGE would have given public health officials additional support in declaring an outbreak and making appropriate public health decisions.

Characterization of spontaneous, In vitro-selected, rifampin-resistant mutants of Mycobacterium tuberculosis strain H37Rv.

Resistance to rifampin in Mycobacterium tuberculosis results from mutations in the gene coding for the beta subunit of RNA polymerase (rpoB). At least 95% of rifampin-resistant isolates have mutations in rpoB, and the mutations are clustered in a small region. About 40 distinct point mutations and in-frame insertions and deletions in rpoB have been identified, but point mutations in two codons, those coding for Ser(531) and His(526), are seen in about 70% of rifampin-resistant clinical isolates, with Ser(531)-to-Leu (TCG-to-TGG) mutations being by far the most common. To explore this phenomenon, we isolated independent, spontaneous, rifampin-resistant mutant versions of well-characterized M. tuberculosis laboratory strain H37Rv by plating 100 separate cultures, derived from a single low-density inoculum, onto rifampin-containing medium. Rifampin-resistant mutants were obtained from 64 of these cultures. Although we anticipated that the various point mutations would occur with approximately equal frequencies, sequencing the rpoB gene from one colony per plate revealed that 39 (60.9%) were Ser(531) to Leu. We conclude that, for unknown reasons, the associated rpoB mutation occurs at a substantially higher rate than other rpoB mutations. This higher mutation rate may contribute to the high percentage of this mutation seen in clinical isolates.

Diversity and prevalence of PorA types in Neisseria meningitidis serogroup B in the United States, 1992-1998.

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.

An analytical model applied to a multicenter pneumococcal enzyme-linked immunosorbent assay study.

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.

Coccidioidomycosis in human immunodeficiency virus-infected persons in Arizona, 1994-1997: incidence, risk factors, and prevention.

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.

Risk factors for acute symptomatic coccidioidomycosis among elderly persons in Arizona, 1996-1997.

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.

Risk factors associated with leptospirosis in northeastern Thailand, 1998.

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.