Capture-recapture methodology, originally developed for estimating demographic parameters of animal populations, has been applied to human populations. This tutorial reviews various closed capturerecapture models which are applicable to ascertainment data for estimating the size of a target population based on several incomplete lists of individuals. Most epidemiological approaches merging di erent lists and eliminating duplicate cases are likely to be biased downwards. That is, the ÿnal merged list misses those who are in the population but were not ascertained in any of the lists. If there are no matching errors, then the duplicate information collected from a capture-recapture experiment can be used to estimate the number of missed under proper assumptions. Three approaches and their associated estimation procedures are introduced: ecological models; log-linear models, and the sample coverage approach. Each approach has its unique way of incorporating two types of source dependencies: local (list) dependence and dependence due to heterogeneity. An interactive program, CARE (for capture-recapture) developed by the authors is demonstrated using four real data sets. One set of data deals with infection by the acute hepatitis A virus in an outbreak in Taiwan; the other three sets are ascertainment data on diabetes, spina biÿda and infants' congenital anomaly discussed in the literature. These data sets provide examples to show the usefulness of the capture-recapture method in correcting for under-ascertainment. The limitations of the methodology and some cautionary remarks are also discussed.A. CHAO ET AL. ascertained by three sources: (i) P-list, records based on a serum test taken by the Institute of Preventive Medicine, Department of Health of Taiwan -there were 135 identiÿed cases; (ii) Q-list, local hospital records reported by the National Quarantine Service -122 cases were found; (iii) E-list, records collected by epidemiologiststhere were 126 cases. Merging the three lists by eliminating duplicate records resulted in 271 ascertained cases. This data set has the advantage of a known true number of infected because a screen serological check for all students was conducted after the three surveys. In Section 5, we use this data set to show the need of correction for undercount. 2. Hook et al. [2] and Regal and Hook [3] presented a data set on spina biÿda collected in New York State from 1969-1974. Three lists were collected from birth certiÿcates, death certiÿcates and medical rehabilitation ÿles. There were 513, 207 and 188, cases, respectively, on the three lists and in total 626 ascertained cases. A method of estimating the number of missed cases was discussed by the above authors to assess the completeness of the survey and to accurately estimate the prevalence rate. 3. Bruno et al. [4] collected a data set on diabetes in a community in Italy based on the following four records: diabetic clinic and=or family physician visits (1754 cases); hospital discharges (452 cases); prescriptions (1135 cases), and pu...

. This assay had a specificity of 100% and various sensitivities of at least 3.5 PFU/ml for YFV, 2.0 PFU/ml for JEV, 10.0 PFU/ml for WNV, and 10.0 PFU/ml for SLEV. Additionally, we have developed an in vitro transcription system to generate RNase-resistant RNA templates for each of these eight viruses. These templates can be incorporated into the assay as RNA copy number controls and/or as external controls for RNA-spiked mosquito pools for quality assurance purposes. Although further study with mosquitoes collected in the field is needed, the incorporation of this assay into mosquito surveillance could be used as an early-warning system for the detection of medically important flaviviruses, particularly when the cocirculation of multiple viruses in the same region is suspected.Flaviviruses are significant causes of disease worldwide and can be classified serologically into several antigenic complexes (12,20). Medically important members of the Japanese encephalitis virus (JEV) serocomplex include JEV, St. Louis encephalitis virus (SLEV), and West Nile virus (WNV). Each of these viruses causes similar disease syndromes in humans, ranging from an asymptomatic or a mild flu-like illness to clinical encephalitis (18). Until 1999, SLEV was the only mosquito-borne flavivirus causing significant human morbidity and mortality in the United States (22). WNV, traditionally found in Europe, Africa, the Middle East, and Asia, emerged in New York City in the late summer of 1999 (1). By 2005 the distribution of WNV had expanded into areas of known recent SLEV activity, such as the states of Florida, Mississippi, Louisiana, Texas, Arizona, Colorado, and California (3). Mosquito surveillance for detection of virus activity during the transmission season is an essential tool for implementation of an effective mosquito control strategy. As WNV and SLEV occupy similar ecological niches in North America, it is critical to develop a cost-effective assay capable of detecting the presence of either one or both viruses simultaneously in the mosquito pools. In addition, dengue virus (DENV) serotypes 1 to 4 (DENV-1 to DENV-4, respectively), SLEV, and yellow fever virus (YFV) are endemic in Latin America (5). The situation is further complicated by the detection of WNV activity in Mexico and Central America (3). Therefore, the development of a real-time, multiplex reverse transcriptase (RT) PCR platform for the simultaneous detection of viral RNA genomes in the Americas is crucial for supporting mosquito surveillance efforts and clinical diagnosis.The RNA-dependent RNA polymerase (RdRp) domain, located at the carboxy terminus of nonstructural protein 5 (NS5), is the most conserved coding region in the Flavivirus genomes (15,16,23). The consensus sequence primers (primers FU1 and CFD2; YFV genomic positions, nucleotide [nt] 8997 and nt 9233, respectively) have been used in our previous study for genetic characterization of this domain for all registered flaviviruses (16). The nucleotide sequences flanked by these two primers were variable...

The use of immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) serves as a valuable tool for the diagnosis of acute flaviviral infections, since IgM antibody titers are detectable early, peak at about 2 weeks postinfection, and subsequently decline to lower levels over the next few months. Traditionally, virus-infected tissue culture or suckling mouse brain (SMB) has been the source of viral antigens used in the assay. In an effort to provide a reliable source of standardized viral antigens for serodiagnosis of the medically important flaviviruses, we have developed a eukaryotic plasmid vector to express the premembrane/membrane and envelope proteins which self-assemble into noninfectious virus-like particles (VLPs). In addition to the plasmids for Japanese encephalitis virus, West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and dengue virus type 2 (DENV-2) reported earlier, we recently constructed the DENV-1, -3, and -4 VLP expression plasmids. Three blind-coded human serum panels were assembled from patients having recent DENV, SLEV, and WNV infections to assess the sensitivity and specificity of the MAC-ELISA using VLPs or SMB antigens. In addition, serum specimens from patients infected with either Powassan virus or La Crosse encephalitis virus were used to evaluate the cross-reactivity of seven mosquitoborne viral antigens. The results of the present studies showed higher sensitivity when using SLEV and WNV VLPs and higher specificity when using SLEV, WNV, and the mixture of DENV-1 to -4 VLPs in the MAC-ELISA than when using corresponding SMB antigens. Receiver operating characteristic (ROC) curve analysis, a plot of the sensitivity versus false positive rate (100 ؊ specificity), was applied to discriminate the accuracy of tests comparing the use of VLPs and SMB antigen. The measurement of assay performance by the ROC analysis indicated that there were statistically significant differences in assay performance between DENV and WNV VLPs and the respective SMB antigens. Additionally, VLPs had a lower cutoff positive/negative ratio than corresponding SMB antigens when employed for the confirmation of current infections. The VLPs also performed better than SMB antigens in the MAC-ELISA, as indicated by a higher positive prediction value and positive likelihood ratio test. Cell lines continuously secreting these VLPs are therefore a significantly improved source of serodiagnostic antigens compared to the traditional sources of virus-infected tissue culture or suckling mouse brain. Members of the genusFlavivirus have an ϳ11-kb, singlestranded, positive-sense RNA genome which translates and encodes capsid (C), premembrane/membrane (prM/M), and envelope (E) structural proteins and seven nonstructural proteins. During natural flavivirus infection, noninfectious viruslike particles (VLPs) are produced in addition to infectious, mature virions (25). Flavivirus VLPs have structural and physiochemical properties similar to mature virus particles. VLPs have been characterized ...