To achieve higher effectiveness in population-based SARS-CoV-2 surveillance and to reliably predict the course of an outbreak, screening, and monitoring of infected individuals without major symptoms (about 40% of the population) will be necessary. While current testing capacities are also used to identify such asymptomatic cases, this rather passive approach is not suitable in generating reliable population-based estimates of the prevalence of asymptomatic carriers to allow any dependable predictions on the course of the pandemic.This trial implements a two-factorial, randomized, controlled, multi-arm, prospective, interventional, single-blinded design with cluster sampling and four study arms, each representing a different SARS-CoV-2 testing and surveillance strategy based on individuals’ self-collection of saliva samples which are then sent to and analyzed by a laboratory. The targeted sample size for the trial is 10,000 saliva samples equally allocated to the four study arms (2500 participants per arm). Strategies differ with respect to tested population groups  and testing approach (without vs. with pre-screening survey). The trial is complemented by an economic evaluation and qualitative assessment of user experiences. Primary outcomes include costs per completely screened person, costs per positive case, positive detection rate, and precision of positive detection rate.Systems for active surveillance of the general population will gain more importance in the context of pandemics and related disease prevention efforts. The pandemic parameters derived from such active surveillance with routine population monitoring therefore not only enable a prospective assessment of the short-term course of a pandemic, but also a more targeted and thus more effective use of local and short-term countermeasures.DRKS00023271. Registered November 30, 2020, with the German Clinical Trials Register (Deutsches Register Klinischer Studien)ClinicalTrials.gov  In 2020, the novel coronavirus SARS-CoV-2 led to the global spread of the coronavirus disease COVID-19), which can cause a highly fatal severe acute respiratory syndrome (SARS). In Germany, SARS-CoV-2 infections first occurred in localized hotspots in early spring, but soon spread exponentially to the larger population, with further spread contained by a 2-month-long nationwide lockdown soon after [9, which A second SARS-CoV-2 wave reached Germany in fall 2020, characterized by more hotspots than in the first wave, and a fast spread of the virus across different population groups. Although inevitable to ensure disease prevention and functionality of the healthcare system, the discussion of a second nationwide lockdown was faced with strong political and societal criticism, which led initially to milder measures with respect to limiting social encounters when compared to the first wave.Germany, as most other countries, relies largely on passive monitoring strategies with regard to SARS-CoV-2 surveillance. This usually includes a monitoring focus on those individuals presenting with clinical COVID-19 symptoms and the tracing of contact persons in case of a positive test result. To effectively monitor virus spread and to reliably predict the course of an outbreak, an active surveillance strategy may improve the monitoring and prediction of the spread of an outbreak. These include monitoring of the entire population, hence identifying and observing also asymptomatic SARS-CoV-2 carriers. Such asymptomatic carriers are estimated to represent about 40% of the population during a pandemic and therefore play a key role in uncontrolled infection spread .In contrast to passive surveillance, active routine surveillance further produces more reliable as well as sufficiently dynamic prevalence estimates for both asymptomatic and symptomatic virus carriers within a defined population. Such dynamic prevalence measures are crucial to not only allowing more accurate predictions on the course of a pandemic, but also in providing policy makers with essential information on containment measures that are more locally tailored and overall, less invasive. The World Health Organization therefore recommends that countries perform active surveillance, including testing, isolating cases, and tracing contacts . FurtherCoV-Surv Study is therefore to evaluate four different active surveillance strategies for their respective effectiveness in determining and predicting the prevalence of SARS-CoV-2 infections in a defined population. To also address any economic considerations, the CoV-Surv Study also assesses the costs and cost-effectiveness of each of the four strategies, compared to the status quo of passive surveillance. In addition, the CoV-Surv Study also includes an additional qualitative component to determine the acceptability of each strategy within the participating population to gain further insight into the overall applicability and user-friendliness of each strategy.This trial therefore intends to generate evidence on the effectiveness and cost-effectiveness of different context-specific pooled sample active surveillance strategies. From the perspective of health policy makers, a key aspect of active population surveillance is to identify the most appropriate strategy with respect to its effectiveness and cost-effectiveness in the context of a health system’s defined testing capacity. The aim of the The novel coronavirus SARS-CoV-2 appeared first in Wuhan, China, in December 2019 and was characterized by the clustered occurrence of viral pneumonia cases . Since tSymptoms of SARS-CoV-2 infection range from fever, headache, and cough to severe shortness of breath with signs of respiratory failure , 9. SARSThe gold standard for laboratory-based SARS-CoV-2 testing includes the analysis of swabs taken from the upper respiratory tract using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), a technology used to detect viral RNA . This RTWith respect to mass testing, the collection of nasopharyngeal or oropharyngeal swabs by medically trained personnel, the required infection protection measures would pose an additional logistical challenge. A more feasible alternative sampling strategy is therefore to have individuals obtain their own swabs and sent them to the laboratory or testing site. There is also increasing evidence that simple saliva, sputum, or throat samples produced from gargling are suitable testing materials with respect to sensitive SRAS-CoV-2 detection –16. FurtCoV-Surv Study will inform the choice of the most effective, acceptable, and cost-effective strategy for SARS-CoV-2 screening and testing, with the most effective and cost-effective strategy expected to be incorporated into the local public health department’s routine health surveillance activities. Additional investigation of its everyday performance will provide insight in the strategy’s applicability to real-time prevalence prediction and the usefulness of the resulting information for local policy makers to implement countermeasures to effectively prevent future nationwide lockdowns.Findings of the Identification of the one best strategy will be determined by a series of parameters. Primary study outcomes include cost per correctly screened person ; cost per positive symptomatic and asymptomatic case ; positive detection rates ; and the precision of positive detection rates .Further, secondary outcomes include the overall participation rate , costs per asymptomatic case, prevalence estimates, number of asymptomatic cases by strategy, ratio of symptomatic to asymptomatic cases by strategy, and participant satisfaction.CoV-Surv Study includes additional study components, i.e., a cost-effectiveness analysis, a prognostic modeling, and a qualitative assessment of participant perception and experience, which will complement trial data with data from other sources to address the following tertiary outcomes: cost-effectiveness of each of the four active strategies compared to the status quo of passive surveillance, the development of a prognostic model to predict hospital utilization caused by SARS-CoV-2, time between test shipment and test application, and time between test shipment and test result, as well as the perception and preferences of persons in respect to being subjected to surveillance.Lastly, the CoV-Surv Study is designed as a two-factorial, randomized, controlled, multi-arm, prospective, interventional, single-blinded trial with cluster sampling and four study arms, each representing a different SARS-CoV-2 testing and surveillance strategy. Figure The trial component of the Individuals (one per household) receive information and study material by mail with instructions on how to produce a saliva sample and how to return the sample by mail. Once received by the laboratory, the sample is tested for SARS-CoV-2 using RT-LAMP.Individuals (one per household) receive information and study material by mail with instructions on how to produce their own as well as saliva samples from each household member and how to return these samples by mail. Once received by the laboratory, the samples are tested for SARS-CoV-2 using RT-LAMP.Individuals (one per household) receive information by mail on how to complete a brief pre-screening questionnaire which asks about COVID-19-related clinical symptoms and risk exposures. Only individuals whose pre-screening score crosses a defined threshold will then receive additional study material by mail with instructions on how to produce a saliva sample and how to return the sample by mail. Once received by the laboratory, the saliva sample is tested for SARS-CoV-2 using RT-LAMP.Individuals (one per household) receive information by mail on how to complete a brief pre-screening questionnaire which asks about COVID-19-related clinical symptoms. Only individuals whose pre-screening score crosses a defined threshold will then receive additional study material by mail with instructions on how to produce their own as well as saliva samples from each household member and how to return these samples by mail. Once received by the laboratory, the samples are tested for SARS-CoV-2 using RT-LAMP.Of the four surveillance strategies, strategy A1 is considered the gold standard for prevalence estimation and thus used to determine the bias introduced by each of the other strategies. To determine the cost-effectiveness, each strategy is compared to a status quo, defined as the currently practiced passive surveillance approach. In each strategy, RT-LAMP-positive samples are additionally analyzed with real-time quantitative RT-PCR (RT-qPCR) in order to minimize the number of false positives.CoV-Surv Study is conducted in the city of Heidelberg and the neighboring Rhein-Neckar district located in the Southwest of Germany. The combined catchment area includes approximately 700,000 inhabitants. During the first pandemic wave in May 2020, the 4-week cumulative SARS-CoV-2 prevalence of symptomatic cases for the study population was about 0.25%.The Eligible are individuals age 7 years or older living in the Rhein-Neckar District or Heidelberg who consented  to provide a self-collected saliva sample  after completion of a brief questionnaire (strategies B1 and B2 only). For the qualitative component complementing the trial, different samples of trial participants and non-participants  are identified for an additional interview. For these interviews, only individuals age 18 years or older are eligible.Participants are asked to provide a saliva sample in a testing tube after gargling with 5ml saline solution. This sample is shipped to the laboratory by mail and then analyzed for SARS-CoV-2 using the RT-LAMP test. All positive-tested samples undergo additional confirmation analysis using the RT-PCR test. Depending on the strategy, some participants first take part in an online pre-screening survey. This short survey contains between 10 and 15 questions assessing the presence of symptoms of an early SARS-CoV-2 infection. This survey also collects information on a participant’s socio-demographic background . Participants of the qualitative study component further take part in a qualitative survey or an interview with open-ended questions. Participation in this qualitative study will commence after an individual’s trial participation has been completed.To estimate the cost of the four surveillance strategies, an activity-based micro-costing approach is used to prospectively estimate the average cost per case completely screened and per case detected for each strategy. Data on the resource consumption of all activities related to the implementation of the four strategies will be categorized as either start-up or implementation cost. For each activity, data on resource consumption for both capital cost items  and recurrent cost items  will be collected directly from respective trial records. Unit costs will be taken from secondary data sources, such as financial statements, invoices, and salary schedules.Participants assigned to strategy A1 are initially contacted by postal mail and receive an information letter outlining and describing the study purpose, the study procedure, the consent processes, and a declaration of consent. This initial letter also contains contact information of the study-specific hotline provided with the support of the local health department. The materials for self-sampling (including a testing tube with a unique barcode identification number) together with a stamped return envelope and instructions on how to return the sample as well as the consent form are also provided in this initial letter. The information letter further contains a link to an online video that provides instructions for the self-sampling procedure. In strategy A1 completion of a short SARS-CoV-2 symptoms survey also provided to each participant is voluntary.Participants assigned to strategy A2 receive in addition to the above testing materials for up to three of their household members and instructions on how to order additional testing materials via the hotline if necessary. The cover letter also includes additional information on the declaration of consent for each participating household member. Completion of the SARS-CoV-2 symptoms survey is voluntary for each household member.Participants assigned to strategy B1 are initially contacted by postal mail and receive an information letter outlining and describing the study purpose, the study procedure, the consent processes, and a declaration of consent. This initial letter also contains contact information of the study-specific hotline provided with the support of the local health department. Prior to receiving testing materials, these participants are asked to consent to and complete an online  SARS-CoV-2 symptom pre-screening survey . A paper-based back-up option for pre-screening is provided to participants without online access. Only participants with a positive result based on the pre-screening will receive testing materials by postal mail.Participants assigned to strategy B2 are in addition to the above asked to conduct the initial pre-screening with all of their household members and will then receive testing material for those individuals in the household with a positive screening result.All tested participants are able to query their test results online using the unique identification number assigned to them. In cases with a positive RT-LAMP test, participants are further informed about the additional RT-qPCR confirmation test. Once this test confirms a SARS-CoV-2 infection, the local health department is informed about the case and the participant will be directly contacted by the health department for additional routine contact tracing.Participants of the qualitative component are contacted either by mail or phone and receive an information letter outlining and describing the purpose of this quality study component and related consent processes.The total study sample consists of 10,000 participants with each strategy enrolling 2500 randomly selected participants. Participants are identified and sampled by each selected municipality’s population register provided by the respective registration office. Individuals or households are drawn as a stratified sample proportional to population size. For each of two strata , individuals or households are drawn in three batches of each three continuous weeks.For the “Rhein-Neckar District” stratum, a two-stage sample design is used for each batch, within which the primary sampling units (PSU) are drawn as synthetic clusters of secondary sampling units (SSU) . First, 55 PSUs are randomly selected for each batch independently using probability proportional to size sampling. These 55 PSUs are then distributed across the 56 municipalities  in the “Rhein-Neckar District” stratum using the Cox algorithm. This might result in some sampling points to contain several PSUs. Second, disproportional sampling is used to draw the same number of SSUs for each sampled PSU in order to obtain a self-weighted sample of individuals or households . Since sThe net sample size is then translated into a corresponding gross sample under consideration of the expected response rates in each strategy and the sensitivity and specificity of the pre-screening tool applied to strategies B1 and B2 (see below). The gross sample therefore includes a total of 21,739 individuals or households contacted in the “Rhein-Neckar District” and 6386 in the “Heidelberg” stratum. This way a sample of 28,125 individuals or households is drawn and then randomly allocated to the four strategies A1, A2, B1, and B2 in the ratio 5 to 2.5 to 14 to 7 to eventually yield a final study sample of 2500 saliva tests for each strategy.For the qualitative component, up to 60 in-depth interviews will be conducted with up to 30 study participants (up to 15 in each of the A and B arms) and with up to 30 participation refusers (up to 15 in each of the A and B arms). Both samples are purposefully selected from the quantitative trial sample to ensure all genders and age groups are represented. These samples serve to explore experiences and perceptions of being subjected to health surveillance and study participation. Further, up to 25 asymptomatic SARS-CoV-2-positive study participants are purposefully selected to explore how asymptomatic men and women diagnosed with SARS-CoV-2 give meaning to their diagnosis and to the dialectic between “feeling healthy” while simultaneously having a high likelihood of transmitting the virus to others. In addition, 100 randomly selected trial participants will be sampled to explore study participants’ perspectives on the implementation of each strategy’s screening and testing processes.The primary analysis will consist of an intent-to-treat (ITT) evaluation. Secondary analysis will include an estimation of the complier average causal effect using standard instrumental variables . Here, tIn order to comprehensively identify the most cost-effective surveillance strategy, the aforementioned ITT analysis is complemented by a cost-effectiveness analysis evaluating the cost-effectiveness of the four surveillance strategies in relation to the status quo of having only passive surveillance. The cost-effectiveness analysis and underlying micro-costing study adopt a health system perspective to account for all costs incurred at the level of the health system, including the economic costs of all resources consumed in relation to the implementation and maintenance of the four active surveillance strategies, as well as the cost of the comparator . All costs are measured as continuous variables for each individual. To determine the cost-effectiveness of each of the four surveillance strategies, costs and effects (measured as the number of cases detected) will be set into relation to those of the status quo. Incremental cost-effectiveness ratios (ICERs) are computed for each surveillance strategy in relation to the status quo. The strategy with the lowest ICER is considered as most cost-effective and will then be further appraised in conjunction with information on the prognostic models (described below) to orient decision making on the choice of the most suitable surveillance strategy to be implemented in routine practice.Prognostic models are based on current SARS-CoV-2-related hospitalization numbers and the test results derived from each strategy using a SEIHR  differential equation. While some parameters for this model are taken directly from the literature, others  are modeled using a Bayesian approach. Given the rather simplified representation of the SARS-CoV-2 infection process in this model, the predictions will only include data within a defined time window to allow models to react more flexibly to changes in the course of the disease. The inference models are adapted to each strategy. Simulations predict each strategy’s ability to determine the time period in which a new outbreak can be detected with an acceptable probability.Based on a dynamic Bayesian prevalence estimate, the successful surveillance strategy is expected to test up to 1000 saliva samples per day in order to generate 2- to 5-week predictions on critical care capacity and other relevant health care parameters in the Rhein-Neckar . Each stStrategy A1 is designed to directly reflect the pandemic events using a purely random sample. With a total of 2500 saliva samples, this strategy is able to detect an overall SARS-CoV-2 prevalence of 0.5% with an accuracy of ±0.35% and a power of 95%; a prevalence of 0.2% would be detected with an accuracy of ±0.25% and a power of 93%  . AssuminSample sizes are therefore designed to yield 2500 saliva samples available for analysis in each study arm. The catchment area of the study  has approximately 700,000 inhabitants. To ensure sufficient precision of the measured estimates, a total net case number of 1.4% of the target population  is targeted. Based on assumptions on participation rates as well as the sensitivity and specificity of the pre-screening tool, the following gross sample sizes are needed for each strategy:For strategy A1, a response rate of 50% is assumed after one-time prompting, warranting a gross sample of at least 5000 addresses per batch; for strategy A2, an average household size of two with a response rate of 50% after prompting is assumed, warranting a gross sample of at least 2500 addresses per batch . For strCoV-Surv Study is expected to identify the most effective, acceptable, and optimal surveillance strategy from multiple scientific perspectives, in order to ensure that the most promising strategy is then to be converted into a routine system as needed. This routine system should be scalable to up to 1000 saliva samples per day to allow reliable predictability during the course of a SARS-CoV-2 wave. For its long-term application, meaning once the SARS-CoV-2 pandemic should be successfully controlled and no longer poses an imminent public health risk, the routine system resulting from the CoV-Surv Study is further expected to become a stand-by surveillance method that can be quickly reactivated any time in the event of new pandemic threats. As surveillance is based on RT-LAMP testing of biological samples, this test can be quickly adjusted to any other pathogen of public health concern.Once completed, the Systems for active surveillance of the general population will gain more importance in the context of pandemics and related disease prevention efforts. As seen during the current SARS-CoV-2 pandemic, the epidemiological course of an easily transmissible illness follows a typical pattern with an initially low overall prevalence in the general population and a comparatively higher prevalence in local clusters. Even in the absence of so-called super-spreader events, infections continue spreading within the general population and therefore lead to increasing overall prevalence rates over time. While complete lockdowns and similarly drastic interventions are effective in reducing the overall prevalence, these measures should only be used over relatively shortly defined time periods. While milder containment methods  effectively prevent local outbreaks, they are usually less effective in preventing infection spread.While useful in identifying positive cases and tracing their potential contacts, passive surveillance strategies should be supplemented by active routine monitoring of larger population groups based on regular testing. Especially in instances where public life is reopened after a more drastic lockdown, active testing frequencies will need to be increased enormously to prevent larger infection chains or super-spreader events to recur, which otherwise would lead to the next lockdown. The pandemic parameters derived from such active surveillance with routine population monitoring therefore not only enable a prospective assessment of the short-term course of a pandemic, but also a more targeted and thus more effective use of local and short-term countermeasures.An ideal active surveillance strategy would frequently test every single person in order to identify all asymptomatic and symptomatic carriers as early as possible and to fully prevent further spread. Obviously, such an ideal strategy is unrealistic and active surveillance approaches therefore will have to work with time-varying randomized samples. Estimated prevalence rates will therefore differ in their uncertainty depending on sample sizes used. This will have significant implications on pandemics like the current one. Since COVID-19 is a respiratory disease that is transmitted similarly to most seasonal viral diseases, SARS-CoV-2 infection rates are also likely to increase during winter months. This is expected to lead to an increase in symptoms similar to COVID-19 during these so-called flu seasons, which will further require the density and frequency of SARS-CoV-2 tests will have to be increased to maintain active surveillance.The final protocol version is “Surveillance_Studienprotokoll_03Nov2020_v1_2” from November 3, 2020. Recruitment started November 18, 2020, and has been completed on December 22, 2020. This protocol was published as a structured summary (https://doi.org/10.1186/s13063-020-04982-z).


CapZ is associated with membranes in cells and it is generally assumed that this interaction is mediated by polyphosphoinositides (PPI) particularly PIPi.e., alpha- and beta-subunits. However, a beta-subunit segment between residues 134–151 is most likely to be involved in interacting with and inserting into lipid membrane due to a slighly higher ratio of positively to negatively charged residues and also due to the presence of a small hydrophobic helix.We propose that non-PPI lipids also bind CapZ. Data from computer-aided sequence and structure analyses further suggest that CapZ could become partially buried in the lipid bilayer probably under mildly acidic conditions, in a manner that is not only dependent on the presence of PPIs. We show that lipid binding could involve a number of sites that are spread throughout the CapZ molecule CapZ may therefore play an essential role in providing a stable membrane anchor for actin filaments. It is regulated by actin-binding proteins that are controlled by a variety of signalling molecules including the well-characterized polyphosphoinositides (PPIs). One of the capping proteins is the calcium-insensitive CapZ, which is regulated by phosphatidylinositol 4,5 bisphosphate (PIP2) -4. This Drosophila, Saccharomyces, Dictyostelium, Acanthamoeba, Caenorhabditis and vertebrates. The protein is composed of two subunits, labelled alpha and beta. The alpha-subunits range between 32 kDa and 36 kDa; the beta-subunits are generally smaller, ranging between 28 kDa and 32 kDa. To date, actin binding has only been ascribed to the beta-subunit [CapZ proteins have been isolated from various species, and sequence studies demonstrate extensive homology among -subunit , althoug-subunit .Spatial and temporal localization studies in non-muscle cells have not always produced a consistent picture: in one case the distribution is nuclear, while chicken CapZ is concentrated in epithelial cell-cell junction complexes. Yeast capping proteins are found at the membrane in regions generally rich in actin . In musc2) and could therefore interact with, or embed into, lipid regions consisting of phospholipids, glycolipids, cholesterol and/or long-chain fatty acids. Our computational analysis indicates that the C-terminal half of CapZ beta-subunit could contribute to lipid interaction/insertion. CapZ may therefore play an essential role in providing a stable membrane anchor for actin filaments.Here we report that CapZ has the potential to bind to lipids  and rtm =<H>/0.51. Three conditions exist, depending on the Eisenberg plot [rsurface and rtm are both less than or equal to 1.0, then the polypeptide region is globular; (2) if either rsurface or rtm is greater than 1.0 and the other less than or equal to 1.0, then the larger ratio determines the characteristic property; (3) if both values are greater than 1.0, then the region is said to be surface-seeking.To detect lipid membrane binding and hydrophobic motifs, and potentially antigenic regions, a window size of 11 residues was employed. The algorithm for detecting putative lipid-binding hydrophobic polypeptide sequence segments discriminates between surface-seeking and transmembrane regions. Computationally, this is performed by constructing and interpreting plots for the average hydrophobicity <H> and the average hydrophobic moment <μs' scale . Accordirage <H> . In thiset al. [An amphipathic helical region was defined by the simple requirement for an effective interaction between an alpha-helix and acidic lipids. The interaction motif is suitable for amino acid segments with a length of 18 residues, which would represent five complete turns of an ideal alpha-helix. When projected on to a plane, the consecutive residues of an ideal helix are spaced with a periodicity of 3.6 at 100 degree intervals. For the amphiphatic helical analysis, a matrix incorporating information about the distribution of physico-chemically different residues was employed. This matrix also included information regarding amphiphatic structure. This approach is based on a previous treatment by Hazelrig et al. . With anHomo sapiens. The lipid-binding properties of each candidate site can subsequently be evaluated using a variety of in vitro techniques.Hydrophobic moments of alpha-helices and beta-strands were calculated, assuming periodicities in the hydrophobicity of 3.6 and 2.0 residues, respectively. The entire process yields several candidate sites that relate to sequence and conformational motifs for each candidate protein sequence. The two protein sequences used were obtained from the NCBI database: residues 1 to 286 from the alpha-subunit from NP006126, and residues 1 to 272 from the beta-subunit from NP004921, both from Homo sapiens CapZ correlated with regions in the high-resolution crystal coordinates obtained from Gallus gallus and deposited in the Protein Data Bank (PDB code 1IZN). Over the range of sequences used there was almost 100% identity between the CapZ subunits from Homo sapiens and Gallus gallus. Molecular visualisation software packages, SPDBV and PYMOL, were used to characterize the secondary and tertiary structure, the solvent accessibility and the electrostatic field potentials [Here, the experimentally-supported lipid-binding sites for tentials ,16. ElecThe secondary structure analysis of the CapZ sequence was started with the search for segments with maximum hydrophobic and amphipathic character. The most hydrophobic segments and the most amphipathic helical segments were found in the amino-terminal region of the protein between residues 113–130 and 225–242 both in the alpha-subunit and between residues 134–151 and 215–232 both in the beta-subunit.rtm and rsurface ratio profiles evaluate the hydrophobic or amphipathic alpha-helical stretches. For these calculations an amino acid window size of 11 was used. The plot in (c) represents the matrix calculations for an amphipathic alpha-helix motif. At a window size of 18 residues, the consensus score of the existing sequence (continuous line) and the average consensus score of 400 sequence randomizations (dotted line) are plotted for every segment. For any segment, the standard deviation (SD) of the randomizations is denoted by a vertical bar in the SD, where factor Γ was greater than 3.0. The quantitative distribution of charged amino acids within 7-residue segments in (d) are marked by the continuous and discontinuous lines of positively and negatively charged residues.Figures Results from the plots in Figures Specifically, the segment 113–130 in the alpha-subunit shows a high ratio of positively and negatively charged amino acids that form the hydrophilic side of the amphipathic helix. The hydrophobic helix shows seven non-polar and three polar amino acids and would be poorly-equipped for lipid binding/insertion. The segment 225–242 in the alpha subunit, however, shows high contents of positively and negatively charged and polar amino acids, and could interact strongly with the hydrophilic (and hydrogen-bonding) side of the opposite amphipathic helix. The hydrophobic side of the helix contains six non-polar and one polar amino acid, including a strongly hydrophobic amino acid . This gives this helix its predominantly amphipathic character. The glutamic acids (deprotonated at pH 7.0) at positions 11 and 13 would seem to make the helix unsuitable for surface binding to a negatively-charged lipid layer.The segment 134–151 in the beta-subunit shows a slightly higher ratio of positively to negatively charged amino acids on the hydrophilic side of the short amphipathic helical region within the beta-strands, whereas the hydrophobic helical side contains seven non-polar and one polar amino acid. This distribution of positively charged amino acids would be more favourable for surface binding to negatively charged lipid layers. The segment 215–232 in the beta-subunit shows a similar amphipathic charge distribution to segment 225–242 in the alpha-subunit; however, the (negatively charged) glutamic acid at position 7 probably makes any surface binding to lipid unfavourable.via lysine 136).The recent crystal of CapZ shows two subunits that are structurally analogous creating a pseudo-two-fold symmetry perpendicular to the long axis of the molecule Figure . Each su2 molecules [2 has been contentious, complicated by differences between studies in the state or presentation of the lipid. However, when presented as a minor component with other lipids (i.e. cholesterol), one PtdInsP2 binds one gelsolin, close to the physiological situation of 0.3–1.5%, which then allows it to associate with the plasma membrane [Recently, it has been reported that when gelsolins  are presented with high lipid concentrations they can bind as many as ten PtdInsPolecules . The valmembrane .2, if they are available, through direct hydrogen-bonding interactions with the binding sites; however at lower PtdInsP2 concentrations these sites may be occupied by other lipids. This is in agreement with observations by differential scanning calorimetry, film balance and spectroscopy, which have shown that proteins require a net negative charge created by lipids other than PPIs, a hydrophobic interface or indeed PPI for membrane interaction/insertion [Furthermore, it has been reported that polyphosphoinositides (PPI) form aggregates within the bilayer under the influence of certain proteins  and thernsertion .2 has been assumed to be the binding partner of CapZ and yet this lipid dissociates the CapZ-actin complex [CapZ has been found to be associated with both membranes and actin filaments in activated macrophages and platelets ,22. This complex ,24. It ietc. have this capacity in isolation [This work raises the possibility that CapZ not only binds to the lipid surface, but also becomes partially embedded within the lipid bilayer due to the residues 134–151 of its beta-subunit. Previous studies have indicated that various peptides derived from PPI-binding regions of, for example gelsolin, Arp2/3, talin solation . The autsolation . The impsolation .In conclusion, a number of sites in CapZ have been proposed to bind lipids and these tend to be located in linker regions between the discrete domains of the protein. The main sites appear to be in the linker regions, 134–151 and 215–232 in the beta-subunit and secondary sites have been identified within the alpha-subunit. We suggest further that the first region 134–151 in the beta-subunit becomes inserted between lipid heads and perhaps into the core of a lipid bilayer.


Moreover, using average iteration and abundance scores for the 12 phenotypes and sex as their relevancy measurements, we found all relevant phenotypes for all four populations except phenotype b for the Karangar population, with suggested subgroup structure consistent with latent traits used in the model. In conclusion, our findings suggest that the ADTrees method may offer a more accurate representation of the disease status that allows for better detection of linkage evidence.We applied the alternating decision trees (ADTrees) method to the last 3 replicates from the Aipotu, Danacca, Karangar, and NYC populations in the Problem 2 simulated Genetic Analysis Workshop dataset. Using information from the 12 binary phenotypes and sex as input and Kofendrerd Personality Disorder disease status as the outcome of ADTrees-based classifiers, we obtained a new quantitative trait based on average prediction scores, which was then used for genome-wide quantitative trait linkage (QTL) analysis. ADTrees are machine learning methods that combine boosting and decision trees algorithms to generate smaller and easier-to-interpret classification rules. In this application, we compared four modeling strategies from the combinations of two boosting iterations  coupled with two choices of tree generation types . These four different strategies were applied to the founders in each population to construct four classifiers, which were then applied to each study participant. To compute average prediction score for each subject with a specific trait profile, such a process was repeated with 10 runs of 10-fold cross validation, and standardized prediction scores obtained from the 10 runs were averaged and used in subsequent expectation-maximization Haseman-Elston QTL analyses (implemented in GENEHUNTER) with the approximate 900 SNPs in Hardy-Weinberg equilibrium provided for each population. Our QTL analyses on the basis of four models  detected evidence for linkage (Z ≥ 1.96,  Alternating decision trees (ADTrees) are machine learning methods combining boosting and decision trees algorithms to generate classification rules . TraditiADTrees, a natural extension of both voted-stumps and decision trees, consist of alternating layers of prediction and decision nodes . The strt, a decision node, together with its two prediction nodes, is introduced. For full ADTrees, the decision node may be attached to any previous prediction node, leaf nodes or otherwise, including the root prediction node. Each prediction node is associated with a weight, α, which represents its contribution to the final prediction score, F(x), for every path that reaches it. Hence the contribution of each decision node may be understood in isolation, and summing the individual contribution gives rise to the final prediction and classification.Boosting is a general and effective method of combining moderately successful rules to produce very accurate prediction. As outlined in Figure In this study, we present the results of ADTrees in conjunction with genome-wide quantitative trait linkage (QTL) scans. Three quantitative measurements, including prediction, iteration, and abundance scores, were obtained from the ADTree-based classifiers using information from the 12 phenotypes and sex. With the help of the quantitative information, we aimed to gain a better understanding of the relationship between disease phenotypes and affection status as well as the risk profiles for each individual. The prediction scores, serving as a composite view of disease status, were then used to carry out genome-scan linkage analysis.The Problem 2 simulated data were used in this study and all analyses were performed without knowledge of the answers except the final comparison with disease models and regions of real linkage. The datasets comprised four populations, Aipotu, Danacca, Karangar, and NYC. Within each population, we arbitrarily selected the last three replicates out of the total 100 replicates available to the Genetic Analysis Workshop 14 (GAW14) participants.We used ADTrees decision-trees with AdaBoost boosting algorithm implemented in MLJAVA  to constWe chose founders in each population to construct the classifiers. The classifiers for each population were trained and tested by a total of 10 runs of 10-fold cross validation. Within each run, one-tenth of the founders in each population were reserved as a test set and the rest were used as a training set.The prediction score of disease status for each individual was derived directly from the ADTree model. Figure The iteration score and abundance score were additional quantitative measurements estimated from the individual tree structure associated with a classifier built during each run . The numThe prediction scores were used as a quantitative trait for QTL analysis in four populations using 917 single-nucleotide polymorphism (SNP) genotypes in the last 3 replicates. Prior to the analysis, Hardy-Weinberg equilibrium (HWE) tests of each population were first carried out using founder genotype information. Around 900 SNPs in HWE  were then retained for further analyses. Four genome scans for each population were performed using prediction scores computed from four different classification models; that is, full-ADT or BDT tree models paired with either exponential or log loss function. The expectation-maximization Haseman-Elston (EM-HE) method as implemented in GENEHUNTER [In all four populations, the average size of iteration steps over 10 runs of 10-fold cross validation during the boosting appeared to be the smallest in the full-ADT and largest in the BDT classifiers, suggesting that the full-ADT classifier performed better than the BDT. Although the log loss function appeared to perform slightly better than the exponential loss function except in the Karangar population, the two loss functions only differed by one step. The average size of all four classification models in all four populations is presented in Table Full-ADT with either exponential or log loss function extracted all relevant phenotypes used in the disease simulation models for all four populations without any false positive through either iteration or abundance scores averaged over 10 runs of 10-fold cross validation, as shown in Table Furthermore, iteration scores of relevant phenotypes from full-ADT suggested certain grouping structures, as shown in Table p < 0.01) on chromosomes 1, 3, 5, and 9, where SNP markers were closest to disease-related loci, as shown in Table Using average prediction scores from full-ADT as a quantitative trait for QTL analysis, with either exponential or log loss function then averaged over 10 runs of 10-fold cross validation, our genome scans attained significant evidence of linkage (Z ≥ 1.96, In this study, we applied the ADTrees method to the Problem 2 dataset and generated a new quantitative trait for each individual based on average prediction scores using information from the 12 phenotypes and sex. In short, the full-ADT approach performed better than BDT, while the exponential and logarithmic loss functions provided essentially comparable results. Using average iteration and abundance scores for the 12 phenotypes and sex, full-ADT extracted all relevant phenotypes without any false positive for all four populations except phenotype b for the Karangar population. Furthermore, iteration scores of relevant phenotypes from full-ADT suggested a subgroup structure consistent with latent traits used in the model. The order of relevant phenotypes by average iteration and abundance scores also indicated that the four populations were quite different from one another. In the QTL analyses, these methods identified significant evidence of linkage where SNP markers were closest to disease-related loci. SNP markers with false-negative and false-positive evidence of linkage were also considered. Future QTL analysis in combination with iteration scores and abundance scores may yield even better results than traditional qualitative trait linkage through a summary of relevant traits that contribute to disease status. In summary, our findings suggest that the ADTrees method is feasible and offers the advantage of a more accurate representation of the disease status.ADTrees: Alternating decision treesBDT: Boosting decision treeEM-HE: Expectation-maximization Haseman-ElstonGAW14: Genetic Analysis Workshop 14HWE: Hardy-Weinberg equilibriumQTL: Quantitative trait linkageSNP: Single-nucleotide polymorphismK-YL conceived of the study, participated in its design, carried out ADTrees analyses, and drafted the manuscript. JL participated in its design, and carried out QTL linkage analyses. XZ was responsible for the background in machine learning methodology. STCW participated in the design of the study. All authors read and approved the final manuscript.


A primate-specific exon is found to be dependent on RNA editing for its exonization. Alu retroelements are specific to primates and abundant in the human genome. Through mutations that create functional splice sites within intronic Alus, these elements can become new exons in a process denoted exonization. It was recently shown that Alu elements are also heavily changed by RNA editing in the human genome.Alu-exon that exclusively depends on RNA editing for its exonization. We demonstrate that RNA editing regulates the exonization in a tissue-dependent manner, through both the creation of a functional AG 3' splice site, and alteration of functional exonic splicing enhancers within the exon. Furthermore, a premature stop codon within the Alu-exon is eliminated by an exceptionally efficient RNA editing event. The sequence surrounding this editing site is important not only for editing of that site but also for editing in other neighboring sites as well.Here we show that the human nuclear prelamin A recognition factor contains a primate-specific Alu sequences can be recruited as a mechanism supporting the birth of new exons in the human genome.Our results show that the abundant RNA editing of  Analysis of the sequenced human genome has revealed that it contains about 200,000 exons . HoweverAlu elements use this exonization mechanism to give rise to hundreds of novel internal exons in the human genome [Alu elements are unique primate-specific retrotransposons that occur in over one million copies in the human genome [Alu-derived internal exons are almost always alternatively spliced, allowing the original isoform to coexist with the new one and preventing the deleterious effects of introducing a new protein at the expense of the original one [Alu elements can increase the coding capacity of human genes while maintaining the original protein repertoire.We have recently shown that n genome . Alu elen genome ,6,7. Then genome ,8,9. Aluinal one ,8. Thus,Alus were reported to contribute to human transcriptome diversity by an additional mechanism, involving adenosine-to-inosine (A-to-I) RNA editing. A-to-I RNA editing refers to the deamination of selected adenosine residues, altering the nucleotide sequence of RNA transcripts from that encoded by genomic DNA. It is catalyzed by enzymes from the ADAR (adenosine deaminase acting on RNA) family. Editing targets are typically located within double stranded RNA (dsRNA), which is recognized by the ADAR enzymes [Alu sequences. Due to the abundance of Alu elements in the human genome, two Alus in opposite orientation are frequently found near each other. When transcribed in the pre-mRNA, these two Alus can presumably fold to create a dsRNA; this substrate is recognized and edited by the ADAR enzymes. Edited Alus typically do not contribute to the protein repertoire, but rather reside in non-coding parts of the pre-RNA molecule - untranslated regions (UTRs) and introns [Recently,  enzymes . RNA edi enzymes ,11,12. T introns -18.Alu elements are both extensively edited and can give rise to novel alternatively spliced exons in primate genomes raises the question of whether RNA editing can be involved in the birth of new Alu-exons. RNA editing was previously shown to regulate alternative splicing by creating a splice site [ADAR2 gene, in which intronic AA dinucleotide turns into a functional AG 3' ss following RNA editing [Alu elements, thus promoting their exonization [Alu-exon that exclusively depends on RNA editing for its exonization. We show that RNA editing regulates the exonization in a tissue-dependent manner, both through creation of a functional AG 3' ss, elimination of a premature stop codon, and regulation of the inclusion/skipping level through alteration of exonic splicing enhancers and silencers within the exon. We also demonstrated that the sequence around an editing site is important not only for the editing in that site but also editing in neighboring sites located along this Alu-exon. Our results show that RNA editing can be recruited as a mechanism supporting the birth of new exons in the human genome.The observation that ice site . The mos editing . Indeed,nization . In thisAlu-derived exons were fixed into protein-coding genes through an RNA-editing-mediated process, we first used expressed sequence tags (ESTs) and cDNAs from GenBank version 136 aligned to the human genome (version hg16) to identify internal human exons that contain Alu elements, as described in [Alu-containing exons flanked by either AA at the 3' ss or AT at the 5'ss. These non-canonical splice sites will normally not be selected by the splicing machinery; however, each of these splice sites is theoretically capable of becoming a bona fide splice site through A-to-I RNA editing, because inosine is recognized by the splicing apparatus as a guanosine [Alu-exon will be supported by more than one EST/cDNA, and that it neither induced frame-shift nor contained a stop codon, as these parameters were shown to be indicative of functional alternatively spliced exons [To check the possibility that ribed in . We lookuanosine . We demaAlu-derived exon, having an AA 3' ss, that conformed to the above conditions . The exon inserts 46 in-frame additional amino acids into the coding sequence of NARF.We were able to detect one such s Figure . This exAlus in human exons, there is a nearby  intronic Alu counterpart in the opposite orientation, which presumably serves as the template for dsRNA formation [Alu sequence 25 bases upstream of the exonized Alu. The antisense Alu has 81% identity when aligned to the exon-producing Alu, suggesting that these two Alus might form a stable intramolecular dsRNA formation following transcription . It ormation ,14. Inden Figure . This, iAlu elements frequently occurs in more than one position, due to the long RNA duplex usually formed by two oppositely oriented nearby Alus [Alu-exon   Figure , althougWe sequenced all of the exon inclusion PCR products and analyzed the editing frequencies at the five editing sites  by adding puromycine  to 293T cell line did not affect the >97% editing efficiency in site E1 (data not shown). This indicates that the high level of editing in site E1 is not due to elimination of unedited, stop-codon containing mRNAs, but rather is indicative of a high efficiency of editing in that site. Apart from the Q/R site of gluR-B [Alu-exon. It further implies that the exonization of the NARF Alu-exon is functional in the human transcriptome.Usually, editing efficiency is much lower than 100% per site, depending on the expression levels of the ADAR enzymes in the given tissue, the secondary structure of the substrate, or the surrounding sequence. As shown above, position E1 in the NARF f gluR-B , which iAlu-exon. Following transfection of this minigene into 293T cells, total RNA was collected, and the splicing pattern of the NARF minigene was examined by RT-PCR analysis using primers specific to the plasmid cDNA and not the endogenous one . We then tested the effect of serial intronic and exonic mutations on the splicing of the Alu-exon . Combined together, these results demonstrate that the exonization of the Alu-exon 8 in NARF is mediated by RNA editing, and that this mechanism also controls the level of inclusion of this exon in different tissues.When the wild-type minigene was transfected into 293T cells, 23% of the mature mRNAs derived from this minigene represented the exon-inclusion form Figure , lane 1.n Figure , lane 2,u Figure , lane 3.Alu-Alu duplex are involved in the regulation of high efficiency editing at the E1 site, we mutated the two nucleotides surrounding the edited site, as well as the nucleotide in the antisense Alu that is postulated to be opposite to the edited nucleotide within the dsRNA  with another putative ESS (TGGTGG), as predicted by RescueESE [Our results indicate that RNA editing not only enables the exonization of the NARF s Figure . This reon Table . Indeed,escueESE . In addiescueESE ,31). In Alu-exon by altering ESRs within the exon, we serially mutated each of the exonic edited sites from A-to-G, simulating 100% editing efficiency. To examine the effect of the exonic sites only we used a minigene in which an A-to-G mutation mimics 100% editing in the 3'ss, and we also deleted the antisense Alu that affects editing of the exonic sites. As shown in Figure To test the possibility that RNA editing regulates the inclusion levels of the NARF Alu-exon 8 is exonized via RNA editing and that RNA editing is also involved in its tissue-dependent regulation. Previously, RNA editing was implicated in both anti-viral protection and transcript diversity regulation; we now show that editing can also support evolutionary processes such as the birth of new exons. In a recent study, Athanasiadis et al. [Alu exonization events (not including the NARF Alu-exon) that were hypothesized to be regulated by RNA editing; our results provide exemplary confirmation of the validity of these predictions.We have demonstrated that the NARF s et al.  presenteAlu elements become exonized through single base-pair mutations that create functional splice sites within their sequences. Yet in the case of the NARF Alu-exon, exonization strictly depends on RNA editing. This situation provides a simple, yet powerful, way to regulate the levels of exon inclusion in a tissue/developmental stage-specific manner. Since editing levels control the level of Alu-exon inclusion, exon inclusion rates would follow the varying editing levels in different tissues  in the specific tissue. Moreover, we show that editing of two out of the five exonic edited sites affects alternative splicing levels  and the sequence upstream and downstream of it are highly conserved between human and chimpanzee. But in the rhesus macaque there was an insertion of AluY (in the sense orientation) immediately upstream of AluSx (the one that exonized in human), leading the antisense AluSg (the one that forms the dsRNA) to be located 344 nucleotides upstream of the sense AluSx (and not 25 nucleotides upstream as it is in human). In addition, there was an insertion of 8 nucleotides in the sense AluSx in the rhesus macaque as well as a deletion of 44 nucleotides that includes the site used in human as 3'ss . These differences raise the question of whether AluSx in the rhesus macaque exonized at all.Interestingly, the E1 as well as the E5 editing sites in the rhesus macaque (but not in chimpanzee) genome encode 'G', thus presenting only the edited version of the gene in those sites. However, there are differences between the genomic sequence of human and chimpanzee and that of rhesus. The Alu-exon in all tested tissues and cell lines indicates that this exon is a bona fide, fixed functional exon in the human genome that originated from an exapted Alu  [ADAR2 gene, which is an Alu-exon of 120 nucleotides (inserts 40 amino acids). The Alu-exon inclusion isoform does not change the specificity of ADAR2 activity compared to the original isoform (exon skipping) but rather changes the rate of the enzymatic activity [The observed exonization of the NARF unction) . An addiactivity .Alu-exon is the only experimentally verified editing site in the coding region that is primate-specific. It would be interesting, therefore, to understand the function of the Alu-containing NARF isoform in the human transcriptome (as it might be responsible for a primate-specific trait); however, as the function of NARF itself is currently not clear, this must await future studies. A Pfam analysis indicates that the Alu-exon is inserted in NARF within a domain defined as 'Iron only hydrogenase large subunit, carboxy-terminal domain', and hence can presumably affect the substrate binding affinity/specificity, or the catalytic activity, of this domain.Few mammalian ADAR substrates in which editing causes amino acid substitutions have been found so far; the first (and most studied ones) encode receptors that are all expressed in the central nervous system, including subunits of the glutamate receptor superfamily , the serAlu [It is worth noting that several other editing targets that cause predicted amino acid changes were detected in a genome-wide search for editing in Alu ,15, but ADAR2 gene changes, in turn, the editing capacitates of the ADAR protein itself [Our study provides additional verification for the close relationship between splicing and editing, which was demonstrated when physical association between spliceosomal components and ADAR proteins was reported . The actn itself . Editingn itself ,44. ADARn itself . Our resAlu sequences have the potential to undergo exonization [Alu-exon that seemed to be exonized through RNA editing, indicating that such a combination of evolutionary events is relatively rare in the human genome. However, this evolutionary mechanism for the birth of new exons might recur in other genomes. Moreover, this mechanism might allow additional Alu exonizations in the evolutionary future of Homo sapiens and other primate species. As some Alus are still active in the human genome , a noveAlu retroposition and alternative splicing, RNA editing was not originally 'designed' to serve evolutionary purposes; it was rather recruited for this, probably serendipitously. This demonstrates that the creation of genomic novelty can be assisted by numerous molecular biological mechanisms, most of which were originally designed to function in other processes. The dynamism of our genome can, therefore, arise through surprising paths.We have shown that RNA editing can lead to the creation of a new exon in the human genome. Similarly to Alu elements, as described in [Alu-containing exons were identified using blastn analysis against the Alu consensus with a threshold of 1E-10. Alus having AA/GT or AG/AT 3' ss/5' ss, which flank exons in the protein-coding region of the genes, were taken for further analysis. Only Alu-exons supported by multiple cDNAs and not containing stop codons (in the ESTs and cDNA) were further considered. Exons were manually screened to remove false computational predictions.ESTs and cDNAs from GenBank version 136 were aligned to the human genome (version hg16) to identify internal human exons that contain ribed in Alu-contaNM_012336). Each primer contained an additional extension encoding a restriction enzyme sequence. The PCR product of NARF (2.8 kb) was restriction digested and inserted between the KpnI/XhoI sites in the pEGFP-C3 vector, which contains green fluorescent protein .Oligonucleotide primers were designed to amplify (from human genomic DNA) a minigene that contains exons 7, 8, and 9 (and the introns in between) of NARF  . After PCR amplification the reaction was digested with DpnI restriction enzyme  for 1 h at 37°C, 1-3 μl of the reaction was transformed into the Escherichia coli XL-1 strain, and colonies were picked for Mini-prep extraction . All plasmids were confirmed by sequencing.Overlapping oligonucleotide primers containing the desired mutations were used to amplify a mutation-containing replica of the wild-type minigene plasmid, using 2. Cells were grown to 60% confluence, and transfection was performed using FuGENE6  with 1 μg of plasmid DNA. Cells were harvested after 48 h. Total RNA was extracted using TRIzol Reagent , followed by treatment with 1 U of RNase-free DNase . Reverse transcription (RT) was preformed on 2 μg total RNA for 1 h at 42°C using Oligo-dT reverse primer and 2 U of reverse transcriptase avian myeloblastosis virus .293T cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 4.5 g/ml glucose , 10% fetal calf serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 1 U/ml nystatin . Cells were grown on 6-well plates under standard conditions at 37°C with 5% COThe spliced cDNA products derived from the expressed minigenes were detected by PCR using the pEGFP-C3-specific reverse primer and an exon 7 forward primer . Amplification was performed for 30 cycles, consisting of 1 minute at 94°C, 45 s at 61°C, and 1.5 minutes at 72°C. The products were resolved on 1.5% agarose gel and confirmed by sequencing. Band quantification was performed by densitometry scanning of ethidium bromide stained gels, using ImageJ software .For the NMD treatment, cells 48 h post-transfection were subjected to 100 μg/ml puromycin  for 4 h before RNA extraction.Products from RT-PCR or from PCR obtained from commercial cDNAs  were separated by electrophoresis on 1.5% agarose gels. The appropriate PCR product was excised and the DNA was extracted and purified . Direct sequencing from both ends was done using the ABI PRISM . The editing percentage from direct sequencing was calculated as AluSx between human, chimpanzee and rhesus macaque, and also the rhesus macaque sequence of AluSx and its upstream surrounding AluY sequence. Additional data file The following additional data are available with the online version of this paper. Additional data file Two figures showing semi-quantitative PCR analysisClick here for fileAluSx between human, chimpanzee and rhesus macaque, and the rhesus macaque sequence of AluSx and its upstream surrounding AluY sequenceAlignment of Click here for filePrimer sequences used in this researchClick here for file


To present and compare population-based and antenatal-care (ANC) sentinel surveillance HIV prevalence estimates among women in a rural South African population where both provision of ANC services and family planning is prevalent and fertility is declining. With a need, in such settings, to understand how to appropriately adjust ANC sentinel surveillance estimates to represent HIV prevalence in general populations, and with evidence of possible biases inherent to both surveillance systems, we explore differences between the two systems. There is particular emphasis on unrepresentative selection of ANC clinics and unrepresentative testing in the population.HIV sero-prevalence amongst blood samples collected from women consenting to test during the 2005 annual longitudinal population-based serological survey was compared to anonymous unlinked HIV sero-prevalence amongst women attending antenatal care (ANC) first visits in six clinics (January to May 2005). Both surveillance systems were conducted as part of the Africa Centre Demographic Information System.Population-based HIV prevalence estimates for all women (25.2%) and pregnant women (23.7%) were significantly lower than that for ANC attendees (37.7%). A large proportion of women attending urban or peri-urban clinics would be predicted to be resident within rural areas. Although overall estimates remained significantly different, presenting and standardising estimates by age and location  made some group-specific estimates from the two surveillance systems more predictive of one another.It is likely that where ANC coverage and contraceptive use is widespread and fertility is low, population-based surveillance under-estimates HIV prevalence due to unrepresentative testing by age, residence and also probably by HIV status, and that ANC sentinel surveillance over-estimates prevalence due to selection bias in terms of age of sexual debut and contraceptive use. The results presented highlight the importance of accounting for unrepresentative testing, particularly by individual residence and age, through system design and statistical analyses. In sub-Saharan Africa, surveillance of women attending antenatal care (ANC) is often used to measure prevalence and monitor trends in HIV infection. However, when applying ANC-based HIV prevalence estimates to the general population the following biases should be considered: only pregnant women are eligible for testing  ; women wFertility among HIV-infected women in sub-Saharan Africa is lower than in HIV-uninfected women, except in women aged 15–19 years . In younBias due to the purposive selection of ANC facilities should also be considered when applying ANC-based prevalence estimates to a population ,8. Over-Population-based surveys are more representative of the general population than ANC-based surveys as they include non-pregnant and non-ANC attending pregnant women, as well as men. However, limitations exist and of particular concern is the effect of non-response on HIV prevalence estimates.Population-based surveys have been conducted in several sub-Saharan African countries -20, inclIn this paper we present and compare HIV prevalence estimates from both population-based surveillance and ANC sentinel surveillance within an area of sub-Saharan Africa with high contraceptive use and low fertility. We identify and explore differences, and reasons for differences, between the two surveillance systems, with particular emphasis on unrepresentative selection of clinics in ANC sentinel surveillance and unrepresentative testing in population-based surveillance. Where estimates from the two surveillance systems differ we explore methods to reduce these differences.2), and rural areas. The rural population live in scattered homesteads that are not concentrated in villages.The ACDIS is conducted in the rural sub-district of Hlabisa in northern KwaZulu-Natal, South Africa. It covers 435 square kilometres and a total resident population of 85123 (unpublished data as of January 2005). The 11284 homesteads within the area have been enumerated and mapped using a geographic information system (GIS). The area includes a formally designated urban township, peri-urban areas . All individuals eligible for HIV testing were asked for written informed consent and informed about the potential risks to becoming aware of ones HIV status, about how and where HIV test results and post-test counselling may be accessed and, if found positive, how they may be referred to a local clinic for further screening and assessment of eligibility for antiretroviral treatment. The choice to provide a test sample and to access the HIV test result rests fully with the individual.In December 2001, Hlabisa Health sub-district became the first rural district in South Africa to provide antiretroviral drugs for the prevention of HIV mother to child transmission. Between January and May 2005, alongside the Prevention of Mother-to-Child Transmission (PMTCT) programme, venous blood was taken for routine ANC laboratory tests from all women attending first ANC visits at all six government clinics delivering ANC within the ACDIS. Surplus blood from these samples was also used for anonymous unlinked HIV testing. Parity and age was linked to a woman's HIV test result. Results cannot be linked back to the individual as, apart from date of birth, no personal identifiers were collected.For each participant the most likely clinic at which antenatal care was obtained was predicted on the basis of a GIS accessibility model that estimated travel time to the six government clinics within the surveillance area offering ANC. The model took into account the quality and distribution of the road network, barriers to movement and the likelihood of utilising public transport to access care . The sixAs ANC sentinel surveillance does not collect residency information, ANC attendees were proportionally assigned to one of the three residency types  based on the underlying predicted catchment population of the clinic attended. To assess the reliability of the clinic accessibility model in predicting ante-natal attendance we compared the prediction of the model with reported ante-natal clinic usage amongst women ever reporting a pregnancy within the ACDIS.2 values and all confidence intervals presented are at the 95% level. STATA 9.0  was used for univariate and multivariate analyses. To account for unrepresentative testing, population-based and ANC-based HIV prevalence estimates were standardised for age, age and location , and age and clinic catchment by applying the respective prevalence estimates to samples of women adjusted to proportionally match ACDIS population level data on all women aged 15 to 49 as of 1st January 2005. Women reported to the ACDIS during twice yearly fieldworker visits as having been pregnant (regardless of outcome) during the period 1st July 2004 and 30th June 2005, who were also eligible for population-based testing, were identified to assist comparative analyses.Pearson chiUnrepresentative testing by HIV status was analysed by linking records  between the first (July 2003 to December 2004) and second  population-based HIV surveys. The proportion of women with a negative HIV test result in the first survey who also consented to test in 2005 was applied to all women with a first survey test result from whom consent was sought in 2005.Clinic attendance by residency type (where one or both is either predicted or reported) is presented for women eligible for testing in the population-based survey Figure  as well st July 2004 and 30th June 2005. Of the 14476 women eligible for HIV testing in 2005, 85.3% (12351) were contacted and asked to provide a blood sample. This proportion varied by age-group, parity and pregnancy status . Of those eligible, 36.6% (5293/14476) of women agreed to test, and of those contacted 42.9% (5293/12351) tested (Table Ten percent (1444/14476) of eligible women reported being pregnant between 1A definitive HIV test result was available for 99.5% 5266/5293) of women testing. Amongst all women, HIV prevalence was 25.2% , and in pregnant women 23.8% . Prevalence of HIV varied significantly by age-group amongst all women and pregnant women (both p < 0.001), (Table 66/5293 oAn HIV test result was available for 96.9% 1111/1146) of women attending ANC first visits (median parity 1) and both a test result and date of birth available for 95.4% 1093/1146) of women (median age 23 years). Among the 1111 women for whom there was a test result, 12.7% (141) were reported as attending one of four rural-based clinics, 33.8% (375) one of two peri-urban-based clinics and 53.6% (595) the urban-based clinic  of women who refused to test in 2005. Women with a negative HIV test result from the first population-based survey were more likely to test in 2005 than those with a positive result (61.1% (2322/3801) compared to 52.6% (611/1162), p < 0.001). The figures for pregnant women were statistically similar to those for all women. Applying the consent rate of 61.1% to all women with a first survey test result from whom consent was sought in 2005 made little difference, with the adjusted estimate of 26.4% (1426/5392) remaining significantly lower than the overall ANC-based estimate, (both p < 0.001). Prevalence of HIV among women who tested during the first survey but who refused during the second survey was 1.3 times higher than prevalence among women who tested in both (27.1% [551/2030] compared to 20.8% [611/2933]).The results of this paper show population-based estimates of HIV prevalence in women to be consistently lower than ANC-based estimates. Although there are several possible explanations for this difference (discussed below), one possible explanation that should first be considered is unrepresentative testing in the population-based surveyWithin the population-based survey, women in the 25–29 and 30–34 age-groups presented both the highest HIV prevalence estimates and the lowest proportions agreeing to test. Women resident in the urban area were the overall group least likely to consent to test. Prevalence estimates among groups where the proportion of women contacted consenting to test is particularly low should be interpreted with caution. The proportions of women consenting to test are presented as of those contacted and not as of the full eligible population. Therefore, it may be likely that in terms of HIV status those contacted differ to those not contacted. Further analyses are necessary to explore this possible bias.It is likely that within the ACDIS area, where provision of ANC clinics offering PMTCT services is comprehensive , many woA review of 20 national population-based surveys across sub-Saharan Africa suggests non-responders are likely to have a higher prevalence of HIV than responders and, by applying the most extreme scenario to account for such bias, an adjustment factor of 1.34 may be required . Our anaAlthough statistically similar, the overall population-based estimate for women of 25.2%  was lower than the estimate of 27.3%  from the first ACDIS population-based survey  and the The overall ANC-based estimate of 37.7% , although statistically similar, falls mid-way between estimates of 35.3% 95% CIs: 33.7, 36.9%) amongst pregnant women tested within the Hlabisa sub-District PMTCT programme January to June 2005 ,33 and 3.7, 36.9%Modern contraceptive use in South Africa is the highest in Sub-Saharan Africa and fertility the lowest . In contA study based on regional estimates in three sub-Saharan African countries suggested that to convert ANC-based estimates amongst primagravida to all childless women in general populations with high contraceptive use (20%+) an adjustment factor of 0.6 would be necessary . AmongstThe largest differences between the two surveillance systems were in women aged 15–19 years and nulliparous women. That ANC-based estimates were so much higher than population-based estimates in these two groups probably reflects self-selection bias amongst ANC attendees with regards age of sexual debut and non-contraceptive use. Although women aged 25–34 years have the lowest consent rates for population-based testing and highest HIV prevalence, adjusting for unrepresentative testing by age only removes a significant difference within the rural location group.In areas with high contraceptive use ANC-based estimates would be expected to exceed population-based estimates among women whereas, where fertility rates are reduced due to prevalence of HIV infection the opposite is true . AlthougA study of bias in ANC-based surveillance data suggested that an over-estimation of HIV prevalence due to an over representation of urban-based clinics could be mitigated if urban and peri-urban based clinics were shown to be attracting large numbers of women from rural areas . The resDespite supportive evidence for the accuracy of the predictive model for clinic catchments presented both previously ,40 and hIn an area with high ANC coverage  and wherA study in Hlabisa sub-district showed that 91% of homesteads normally utilise the most accessible primary health clinic . HoweverThe findings of this study suggest that where ANC coverage is high, population-based HIV surveillance systems under-estimate HIV prevalence due to unrepresentative testing by HIV status that results in unrepresentative testing by age and residence. The results also suggest that despite evidence of large numbers of women from rural areas  attending urban and peri-urban clinics, in an area with high contraceptive use and low fertility, resulting in selection bias due to age of sexual debut, ANC sentinel surveillance over-estimates prevalence. Understanding how to appropriately adjust ANC sentinel surveillance estimates to represent HIV prevalence in general populations is important since ANC clinics continue to be a relatively cheap and timely source of data.The findings of this study highlight the possible biases inherent to both surveillance systems and suggest that attention should be paid to unrepresentative HIV testing, particularly by age and residency location. Analysing population-based HIV prevalence estimates, and comparing them to ANC-based estimates, also highlights the importance of not assuming population-based estimates equate to a gold standard.The author(s) declare that they have no competing interests.All authors read and approved the final manuscript prior to submission.BDR carried out data-cleaning of ANC dataset as well as the majority of the analyses and writing.JBF coordinated the 2005 population-based HIV survey and 2005 ANC sentinel surveillance, and conducted data cleaning of population-based surveillance data.VH assisted data analyses.FT assisted data analyses.CH assisted data analyses.TB assisted data analyses.KH assisted with the overall approach of the analyses and writing.TW designed the ANC sentinel surveillance and assisted with the structure of the writing.MLN assisted with the overall approach of the analyses and writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Additional information. A word document that presents key messages, statement, ethics, keywords and abbreviations should any of these be needed.Click here for file


We investigated the plasma levels of oxLDL and Lp-PLA2 activity as well as the autoantibody titers against mildly oxLDL in patients with early rheumatoid arthritis (ERA). The long-term effects of immunointervention on these parameters in patients with active disease were also determined. Fifty-eight ERA patients who met the American College of Rheumatology criteria were included in the study. Patients were treated with methotrexate and prednisone. Sixty-three apparently healthy volunteers also participated in the study and served as controls. Three different types of mildly oxLDL were prepared at the end of the lag, propagation and decomposition phases of oxidation. The serum autoantibody titers of the IgG type against all types of oxLDL were determined by an ELISA method. The plasma levels of oxLDL and the Lp-PLA2 activity were determined by an ELISA method and by the trichloroacetic acid precipitation procedure, respectively. At baseline, ERA patients exhibited elevated autoantibody titers against all types of mildly oxLDL as well as low activity of the total plasma Lp-PLA2 and the Lp-PLA2 associated with the high-density lipoprotein, compared with controls. Multivariate regression analysis showed that the elevated autoantibody titers towards oxLDL at the end of the decomposition phase of oxidation and the low plasma Lp-PLA2 activity are independently associated with ERA. After immunointervention autoantibody titers against all types of oxLDL were decreased in parallel to the increase in high-density lipoprotein-cholesterol and high-density lipoprotein-Lp-PLA2 activity. We conclude that elevated autoantibody titers against oxLDL at the end of the decomposition phase of oxidation and low plasma Lp-PLA2 activity are feature characteristics of patients with ERA, suggesting an important role of these parameters in the pathophysiology of ERA as well as in the accelerated atherosclerosis observed in these patients.Rheumatoid arthritis is a chronic inflammatory disease, associated with an excess of cardiovascular morbidity and mortality due to accelerated atherosclerosis. Oxidized low-density lipoprotein (oxLDL), the antibodies against oxLDL and the lipoprotein-associated phospholipase A Rheumatoid arthritis is a chronic inflammatory condition of unknown etiology affecting primarily the synovium, leading to joint damage and bone destruction . RheumatOxidative modification of low-density lipoprotein (LDL) is an important event in the development and progression of atherosclerosis. Oxidized low-density lipoprotein (oxLDL) is present in atherosclerotic lesions of humans and animal models, and promotes atherosclerosis by several mechanisms -9. oxLDL2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase -sn-glycero-3-phosphocholine as a substrate at a final concentration of 100 μmol/l. The reaction was performed for 10 min at 37°C and the Lp-PLA2 activity was expressed as nanomoles of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine degraded per minute per milliliter of plasma [The Lp-PLAf plasma ,27,28. TSerum lipids were determined after 12 hours overnight fast. Total cholesterol, triglycerides and HDL-cholesterol levels were determined on the Olympus AU560 Clinical Chemistry analyzer  as previously described . LDL-chot test for comparisons between baseline and post-treatment values, while one-way analysis of variance followed by the least significant difference post hoc test was used for comparisons between individual groups. Comparison between baseline and post-treatment CRP levels was performed using the Kruskal–Wallis test while CRP levels between individual groups were compared using the Mann–Whitney U test. Correlation between variables was examined using the Pearson's correlation coefficient. We used multivariate logistic regression analysis to calculate the adjusted odd ratios and 95% confidence intervals for the two study groups.Data were expressed as the mean ± standard deviation. Statistical analysis was performed using the paired Student's P value less than 0.05 were retained in the model as independent variables. The variables included in the univariate analysis were the following: age, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, Apo B, Apo A-I, autoantibody titers against oxLDLL, oxLDLP and oxLDLD, plasma Lp-PLA2 activity, HDL-Lp-PLA2 activity (continuous variables) and female gender (dichotomous variable). All statistical analyses were carried out with SPSS 12.0 . In all cases, P < 0.05 was considered statistically significant.Matched groups were constructed taking into account the significant factors as derived from the univariate logistic regression analysis. The model selection used the backward stepwise method (likelihood ratios), and variables at a Fifty-eight patients with ERA and 63 apparently healthy volunteers participated in the study. The clinical and biochemical characteristics of the study population are presented in Table One year of therapy with DMARDs in ERA patients resulted in a significant decrease of the inflammatory markers CRP and the erythrocyte sedimentation rate as well as in the reduction of the disease activity score for 28 joint indices Table . In addi2 and of HDL-Lp-PLA2, compared with controls , compared with controls. The low Lp-PLA2 activity is in accordance with previously published data by our group, indicating that patients with active juvenile rheumatoid arthritis presented with lower plasma Lp-PLA2 activity compared with those with inactive disease or to controls [2 activity is independently associated with ERA. It is well established that the main cellular source of the plasma form of Lp-PLA2 is monocytes, which secrete this enzyme during their differentiation into macrophages [2 is regulated by various factors, including the differentiation state of the cell and the degree of activation by proinflammatory mediators [2 expression by macrophages in vitro [2 activity found in ERA patients could therefore be attributed to the inflammation-induced decrease in the enzyme expression. According to our previously published results, however, another important determinant of the plasma Lp-PLA2 activity is the plasma LDL level [2 in plasma is mainly bound on LDL particles, whereas a small proportion is associated with HDL [The present study shows for the first time that ERA patients exhibit low plasma Lp-PLAcontrols . The prerophages . The celediators ,32. Mostin vitro . The redDL level ,28. Indewith HDL . We may 2 levels from the LDL-cholesterol levels could also explain our results showing that therapy with DMARDs did not affect either the plasma LDL-cholesterol levels or the plasma Lp-PLA2 activity. A factor that could also influence the plasma Lp-PLA2 levels in ERA patients is Lp(a). Indeed, we [2 activity compared with LDL when assayed at equimolar protein concentrations. Importantly, recent results have provided evidence that the Lp(a)-associated Lp-PLA2 may play an important role by degrading oxidized phospholipids that are preferentially sequestered on Lp(a) [2 activity might have influenced the plasma levels of this enzyme in ERA patients since the mean serum levels of Lp(a) in our patients as well as in controls are between 8.6 and 11.2 mg/dl – according to our previously published results, the plasma levels as well as the distribution of Lp-PLA2 between LDL and HDL can be influenced by the presence of Lp(a) only when plasma levels of this lipoprotein exceed 30 mg/dl [The dependence of the plasma Lp-PLAdeed, we  and othedeed, we  have preon Lp(a) . It is u30 mg/dl .L, oxLDLP and oxLDLD) at baseline compared with controls. One year of therapy with DMARDs resulted in a significant decrease of autoantibody titers against all types of oxLDL compared with the respective baseline values, a finding that could be attributed, at least partially, to the repression of the immune system activation due to immunointervention. Importantly, the antibody titers against oxLDLD are independently associated with ERA, thus providing evidence that such types of mildly oxLDL may be implicated in the pathophysiology of ERA. Indeed, previously published results showed that modified LDL with characteristics of minimally modified LDL, but not extensively oxidized LDL, is present in the synovial fluid of patients with rheumatoid arthritis [An important observation of the present study is that ERA patients exhibited higher autoantibody titers against all types of mildly oxLDL  = lipoprotein (a); Lp-PLAThe authors declare that they have no competing interests.ESL wrote the paper and performed the biochemical measurements. ANG participated in the selection of the patients and therapy. ECP contributed to the biochemical measurements and to writing the paper. AIP participated in the statistical analysis and in writing the paper. AAD participated in the selection of the patients and therapy. ADT conceived the idea for the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.