The assumption of no long reporting delays may result in underestimates of US AIDS incidence.

OBJECTIVE: To evaluate the effect of the assumption of no long reporting delays on estimates of AIDS incidence. DESIGN: Reported AIDS cases must be adjusted for reporting delays to estimate AIDS incidence. We compared the adjustments supplied with the Centers for Disease Control and Prevention (CDC) AIDS Public Information Data Set with a set of adjustments that differ with respect to CDC assumption of no long delays. Both methods assume that no changes in delay have occurred throughout the reporting period. METHODS: Probability distributions of reporting delays were calculated from the delay adjustment weights supplied by CDC, and from an alternative method that estimates the probability of long delays from the surveillance data. AIDS incidence estimates from these two approaches were calculated and compared. RESULTS: The CDC adjustments assume that there will be no reporting delays longer than 61 months, whereas the alternative method estimates that 5.9% of case reports will be delayed longer than 61 months. The CDC adjustments result in lower estimates of AIDS incidence and a flattening of the epidemic. CONCLUSIONS: In addition to a 6.2% reduction in total estimated AIDS incidence, the CDC assumption changes the shape of the estimated epidemic. These may result in as much as 4-21% reductions in model estimates of HIV incidence and prevalence.

The major outer membrane protein, CD, extracted from Moraxella (Branhamella) catarrhalis is a potential vaccine antigen that induces bactericidal antibodies.

The major outer membrane protein of Moraxella (Branhamella) catarrhalis, CD, was detergent-extracted from the bacterial cell wall and purified to homogeneity in high yields by a simple process. The purified protein appeared to exhibit immunogenic properties similar to those of native CD exposed on the surface of the bacterium. Antibodies to CD raised in mice specifically bound to intact B. catarrhalis, as determined by flow cytometry analysis. The IgG subclass distributions of anti-CD antibodies in sera from mice immunized with purified CD or with B. catarrhalis were also similar. CD was found to be antigenically conserved among a panel of B. catarrhalis isolates, as demonstrated by the consistent reactivities of mouse anti-CD antisera with a common 60 kDa protein on immunoblots. Furthermore, convalescent sera collected from patients with otitis media due to B. catarrhalis infection were found to be reactive with the CD protein by immunoblotting. Finally, the purified protein induced antibodies in guinea pigs and mice that exhibited in vitro bactericidal activity against the pathogen. Therefore, the native CD outer membrane protein represents a potentially useful antigen for inclusion in a vaccine against B. catarrhalis.

Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: I. Without exogenous activation.

There has been an increasing need in genetic toxicology to progress from strictly qualitative tests to more quantitative tests. This, in turn, has increased the need to develop better quality assurance and comparative bioassay methods. In this paper, two laboratories tested 10 Salmonella mutagens in order to determine the usefulness of selected chemicals as potential reference materials to calibrate the Salmonella assay. If variance within a bioassay is sufficiently low and the rankings of the compounds are of acceptable consistency, the chemicals later could be evaluated for use as standard control compounds, as audit materials, and as standard reference materials for comparative bioassay efforts. The results demonstrated that the chosen chemicals (with the possible exception of dimethylcarbamylchloride) provide such consistent results in the Salmonella mutagenicity bioassay that they can be used for semi-quantitative calibration and as possible bioassay controls, special audit chemicals, and potentially as reference standards in comparative bioassay efforts. Reference standards, whether used as audit materials or in comparative bioassays, must be used concurrently with the test substances of interest; used without bias; used in a standardized, highly controlled bioassay; and be tested across an appropriate dose range. The study also shows that when these compounds are used as reference standards much care must be given to the number and spacing of doses if highly reproducible slope values are to be generated. We recommend use of a pilot test to establish a dose range for definitive tests and the placement of doses for the definitive tests within the first half of the linear dose-response curve. For appropriate comparisons, one should replicate the tests using the defined dose range and analyze the results in a non-biased statistical manner.

Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: II. With exogenous activation.

In order to determine the usefulness of selected chemicals as potential reference materials for calibrating the Salmonella assay, two laboratories tested a series of Salmonella mutagens that require exogenous activation. When the variance for individual substances within a bioassay is sufficiently low and the rankings of those substances are of acceptable consistency, they can later be evaluated for use as standard control compounds, as audit materials, and as standard reference materials for comparative bioassay efforts. The purpose of this project, therefore, was to evaluate the variability in the mutagenic response of potential reference chemicals that require exogenous metabolic activation in the standard plate-incorporation Salmonella mutagenicity assay, and to develop ranking criteria for mutagenic activity based on these data. Ten indirect-acting mutagens were tested in two laboratories using Salmonella typhimurium TA100 and an Aroclor-induced rat liver S9. Each laboratory conducted four definitive testing rounds. A different batch of S9 was utilized for every two rounds. Of the 10 chemicals tested only 2-anthramine had a mean slope value greater than 1000 revertants/micrograms. Three chemicals had slope values between 1000 and 100; and five chemicals had slope values between 100 and 10. The remaining compound, 9,10-dimethyl-1,2-benz[a]anthracene, could not be placed into a single category because it had slope values on either side of 100 revertants per mg. Coefficients of variance were low (i.e., below 25% in most cases). The low variability achieved in this study may be accounted for by two parameters of the study. First, based on Claxton et al. (1991a) and the S9 optimization for three compounds, the amount of S9 was calibrated to a set amount of protein per plate (1.1 mg/plate). Secondly, the 10 test doses were placed in the initial, linear, nontoxic portion of the dose-response curves. The use of ten closely spaced, nontoxic doses allowed for a more accurate estimate of the slope.

An interlaboratory study of an EPA/Ames/Salmonella test protocol.

7 laboratories participated in a collaborative study to evaluate an EPA standard protocol for the Ames test. The study utilized Salmonella typhimurium (strains TA98 and TA100) and 3 metabolic activation levels (0%, 2%, and 10% S9 in the S9 mix). 6 pure chemicals and 2 complex mixtures were tested as coded unknowns. Ability to obtain qualitative results in agreement with published data was less (% agreement) than that reported in an earlier study (% agreement) by de Serres and Ashby (1981) in which each laboratory used its own protocol. The conclusion from analysis of the quantitative data from this interlaboratory Ames study was that both intralaboratory and interlaboratory variations were substantial. Results for the same substance varied by an order of magnitude or more (CV of 115%) when the mutagenic response was measured as the slope of the dose response in revertants/microgram. Taking interlaboratory variation into account, one chemical must be more than an order of magnitude more mutagenic than another (ratio of slopes greater than 10) to have only an even chance of finding a statistically significant difference between the two chemicals at the 5% level. Such large variations must be taken into account when evaluating Ames/Salmonella data.

Undermatching: a reappraisal of performance on concurrent variable-interval schedules of reinforcement.

The extant data for pigeons' performance on concurrent variable-interval schedules were examined in detail. Least-squares lines relating relative pecks and time to the corresponding relative reinforcements were obtained for four studies. The between-study group slopes for time and pecks and five of seven within-study group slopes from individual studies were less than 1.00. This suggested the generality that pigeons respond less to the richer reinforcement schedule than predicted by matching. For pecks, a nonparametric test for distribution of points also supported this concept of undermatching (to the richer reinforcement schedule). In addition, using mean squared error as the criterion, a cubic curve fit the peck proportion data better than any line or other polynomial. This indicates that the relation between peck and reinforcement proportions may be nonlinear.

Modelling of the flow field surrounding tidal turbine arrays for varying positions in a channel.

The modelling of tidal turbines and the hydrodynamic effects of tidal power extraction represents a relatively new challenge in the field of computational fluid dynamics. Many different methods of defining flow and boundary conditions have been postulated and examined to determine how accurately they replicate the many parameters associated with tidal power extraction. This paper outlines the results of numerical modelling analysis carried out to investigate different methods of defining the inflow velocity boundary condition. This work is part of a wider research programme investigating flow effects in tidal turbine arrays. Results of this numerical analysis were benchmarked against previous experimental work conducted at the University of Southampton Chilworth hydraulics laboratory. Results show significant differences between certain methods of defining inflow velocities. However, certain methods do show good correlation with experimental results. This correlation would appear to justify the use of these velocity inflow definition methods in future numerical modelling of the far-field flow effects of tidal turbine arrays.

A meta-analysis of estimates of the AIDS incubation distribution.

Information from 12 studies is combined to estimate the AIDS incubation distribution with greater precision than is possible from a single study. The analysis uses a hierarchy of parametric models based on a four-parameter generalized F distribution. This general model contains four standard two-parameter distributions as special cases. The cases are the Weibull, gamma, log-logistic, lognormal distributions. These four special cases subsume three distinct asymptotic hazard behaviors. As time increases beyond the median of approximately 10 years, the hazard can increase to infinity (Weibull), can plateau at some constant level (gamma), or can decrease to zero (log-logistic and lognormal). The Weibull, gamma and 'log-logistic distributions' which represent the three distinct asymptotic hazard behaviors, all fit the data as well as the generalized F distribution at the 25 percent significance level. Hence, we conclude that incubation data is still too limited to ascertain the specific hazard assumption that should be utilized in studies of the AIDS epidemic. Accordingly, efforts to model the AIDS epidemic (e.g., back-calculation approaches) should allow the incubation distribution to take several forms to adequately represent HIV estimation uncertainty. It is recommended that, at a minimum, the specific Weibull, gamma and log-logistic distributions estimated in this meta-analysis should all be used in modeling the AIDS epidemic, to reflect this uncertainty.

Dilution assay statistics.

A parametric method of statistical analysis for dilution assays is developed in detail from first principles of probability and statistics. The method is based on a simple product binomial model for the experiment and produces an estimate for the concentration of target entities, a confidence interval for this concentration, and an indicator of the quality of the assay called the p value for goodness of fit. The procedure is illustrated with data from a virologic quantitative micrococulture assay used to quantify free human immunodeficiency virus in clinical trials. The merits of the procedure versus those of nonparametric methods of estimating the dilution inducing a 50% response rate are discussed. Advantages of the proposed approach include plausibility of the underlying assumptions, ability to assess plausibility of specific experimental outcomes through their likelihood, and plausibility of confidence intervals.

Genomic fingerprinting of "Haemophilus somnus" isolates by using a random-amplified polymorphic DNA assay.

The random-amplified polymorphic DNA (RAPD) assay was used to generate DNA fingerprints for 16 isolates of "Haemophilus somnus," and one isolate each of "Haemophilus agni," "Histophilus ovis," "Actinobacillus seminis," Pasteurella haemolytica, and Escherichia coli. The RAPD assay differentiated among "H. somnus" isolates, which shared similarity coefficients of 0.46 to 1.00 on the basis of pairwise comparisons of RAPD markers produced with nine random decamer primers. Three virulent encephalitic "H. somnus" isolates exhibited identical banding patterns, suggesting a common clonal ancestry. The RAPD assay clearly distinguished between the "H. somnus"-"H. agni"-"H. ovis" group and the other bacterial species tested. The results of the present study suggest that DNA fingerprinting of "H. somnus" isolates by the RAPD assay could be valuable in revealing subspecific divisions within this largely unexplored species.

Standardization of sensitive human immunodeficiency virus coculture procedures and establishment of a multicenter quality assurance program for the AIDS Clinical Trials Group. The NIH/NIAID/DAIDS/ACTG Virology Laboratories.

An independent quality assurance program has been established by the Division of AIDS, National Institute of Allergy and Infectious Diseases, for monitoring virologic assays performed by nearly 40 laboratories participating in multicenter clinical trials in the United States. Since virologic endpoints are important in evaluating the timing and efficacy of therapeutic interventions, it is imperative that virologic measurements be accurate and uniform. When the quality assurance program was initially created, fewer than 40% of the laboratories could consistently recover human immunodeficiency virus (HIV) from peripheral blood mononuclear cells (PBMCs) of HIV-infected patients. By comparing coculture procedures in the more competent laboratories with those in laboratories who were struggling to isolate virus, optimal conditions were established and nonessential reagents and practices were eliminated. Changes were rapidly introduced into a laboratory when experience dictated that such modifications would result in a favorable outcome. Isolation of HIV was enhanced by optimizing the numbers and ratios of patient and donor cells used in cultures, by standardizing PBMC separation procedures, by using fresh rather than frozen donor PBMCs, by processing whole blood within 24 h, and by using natural delectinated interleukin 2 instead of recombinant interleukin 2 products in existence at that time. Delays of more than 8 h in the addition of phytohemagglutinin-stimulated donor cells to freshly separated patient PBMCs reduced recovery. Phytohemagglutinin in cocultures and the addition of Polybrene and anti-human alpha interferon to media were not important in HIV isolation. The introduction of a consensus protocol based on this information brought most laboratories quickly into compliance. In addition, monthly monitoring has successfully maintained proficiency among the laboratories, a process that is critical for the scientific integrity of collaborative multicenter trials. Problems which might not be appreciated for months are now being resolved early, before data can be compromised unknowingly. This consensus protocol is recommended for any laboratory attempting to isolate HIV for the purpose of standardizing recovery and for accessing virologic endpoints in clinical trials.

Continuous-wave singly resonant optical parametric oscillator based on periodically poled LiNbO(3).

We report a continuous-wave singly resonant optical parametric oscillator (OPO) based on periodically poled lithium niobate. The simple, two-mirror OPO, pumped by a 1.064-microm Nd:YAG laser, had a 2.6-4.5-W threshold and an output of >1.2 W at 3.3 microm and was tuned over 1.45-1.62 microm (signal) and 3.98-3.11 microm (idler). The noise characteristics and the spectral properties of the device are described.

93% pump depletion, 3.5-W continuous-wave, singly resonant optical parametric oscillator.

We report two cw, singly resonant optical parametric oscillator (OPO) configurations based on periodically poled lithium niobate that result in significantly higher efficiency and output power than in previous studies. Using four-mirror OPO cavities and pumping with a 1.064-microm Nd:YAG laser, we observe 93% pump depletion and obtain ~86% of the converted pump photons as useful idler output. The single-beam, in-the-bucket idler output power of 3.55 W at 3.25 microm corresponds to ~80% of quantum-limited performance. We measure and compare the amplitude noise and spectral bandwidth of the two configurations. We also demonstrate >1 W of tunable cw output over the 3.3-3.9-microm spectral range.

2.5-W, continuous-wave, 629-nm solid-state laser source.

We report an efficient, high-power, cw, 629-nm laser source based on a diode-pumped Nd:YAG laser and a periodically poled lithium niobate (PPLN) frequency converter. This device integrates two separate frequency-conversion steps in a single crystal, taking advantage of the ability to fabricate PPLN with nearly arbitrary grating periods and phase-matching temperatures. This device uses a single PPLN crystal that has two grating regions in series. The first region quasi-phase matches a standard optical parametric oscillator process (1064nm?1540nm +3450nm), and the second region quasi-phase matches a sum-frequency process whereby the pump and the signal light make red light (1064nm+1540nm ?629nm). Using a four-mirror ring cavity, we were able to convert 21% of the 1064-nm pump to 629-nm output, yielding 2.5W of red output with 11.8W of input.

Diffusion-bonded stacks of periodically poled lithium niobate.

We constructed diffusion-bonded stacks of periodically poled lithium niobate (PPLN). Such crystals combine the advantages of planar processing used to make PPLN wafers with the power-handling capability of large apertures. We demonstrated an optical parametric oscillator that uses a 3-mm-thick diffusion-bonded stack consisting of three 1-mm-thick PPLN crystals.

Quasi-phase-matched 1.064-microm-pumped optical parametric oscillator in bulk periodically poled LiNbO(3).

We report a quasi-phase-matched optical parametric oscillator, using bulk periodically poled LiNbO(3). The optical parametric oscillator, pumped by a 1.064-microm Q-switched Nd:YAG laser, was temperature tuned over the wavelength range 1.66-2.95 microm. The oscillation threshold of approximately 0.1 mJ was more than a factor of 10 below the damage limit. The LiNbO(3) crystal, fabricated by application of an electric field to a sample with liquid and metal surface electrodes, was 0.5 mm thick with a 5.2-mm interaction length and a quasi-phase-matched period of 31 microm.

Multigrating quasi-phase-matched optical parametric oscillator in periodically poled LiNbO(3).

We report a widely tunable quasi-phase-matched optical parametric oscillator that uses periodically poled LiNbO(3)with a multigrating structure. The device is tuned by translation of the crystal through the resonator and pump beam, with no realignment needed. With a 1.064-microm acousto-optically Q-switched Nd:YAG pumplaser, we produced noncritically phase-matched tunable IR output from 1.36 to 4.83 microm. The threshold was 6 microJ for a 26-mm interaction length. The extraordinary polarization of LiNbO(3) has better IR transmission than does the ordinary polarization, permitting operation at longer wavelengths with d(33) quasi-phase matching than with conventional Type I birefringent phase matching.

Reproducibility of mammographic classifications.

Wolfe's mammographic classification and a percentage classification are statistically evaluated for inter- and intraobserver bias and agreement by seven mammographers with a set of 200 xeromammograms. The results demonstrate significant bias and disagreement with both methods, raising questions about the clinical limitations of these or other mammographic classifications. However, about 90% of the percentage classifications of pairs of readers are within adjacent categories. This suggests that (1) more experience with precisely defined classifications and protocols, (2) the development and application of readily available instructional materials, and (3) studies to identify and evaluate sources of variation in such classifications may eventually lead to acceptable levels of reproducibility.

Multicenter evaluation of quantification methods for plasma human immunodeficiency virus type 1 RNA.

Six procedures for quantifying plasma human immunodeficiency virus type 1 (HIV-1) RNA were evaluated by nine laboratories. The procedures differed in their sample volume and preparation of samples and methods of amplification and detection. Coded samples in a 10-fold dilution series of HIV-1-spiked plasma were correctly ranked by all six procedures. Subsequently, coded duplicate plasma samples from 16 HIV-1-infected patients were tested using a common set of standards. Several HIV-1 RNA procedures were sufficiently reproducible so that an empiric 4-fold change could be viewed as significant. HIV-1 RNA levels in the patients (up to 370,000 RNA copies/mL) correlated with proviral HIV-1 DNA and were inversely correlated with CD4 cell counts; HIV-1 RNA assays were more sensitive than plasma viremia, standard p24 antigen, or immune complex-dissociated p24 antigen assays. This study demonstrated that several HIV-1 RNA quantitative assays are ready for use in clinical trials.