Chronic neck pain is a common complaint in the Netherlands with a point prevalence of 14.3%. Patients with chronic neck pain are often referred to a physiotherapist and, although many treatments are available, it remains unclear which type of treatment is to be preferred.The objective of this article is to present the design of a randomised clinical trial, Ephysion, which examines the clinical and cost effectiveness of behavioural graded activity compared with a physiotherapy treatment for patients with chronic non-specific neck pain.Eligible patients with non-specific neck pain persisting longer than 3 months will be randomly allocated to either the behavioural graded activity programme or to the physiotherapy treatment. The graded activity programme is based on an operant approach, which uses a time-contingent method to increase the patient's activity level. This treatment is compared with physiotherapy treatment using a pain-contingent method.Primary treatment outcome is the patient's global perceived effect concerning recovery from the complaint. Global perceived effect on daily functioning is also explored as primary outcome to establish the impact of treatment on daily activity. Direct and indirect costs will also be assessed. Secondary outcomes include the patient's main complaints, pain intensity, medical consumption, functional status, quality of life, and psychological variables. Recruitment of patients will take place up to the end of the year 2004 and follow-up measurement will continue until end 2005. Neck pain is a common complaint that causes substantial morbidity in western countries with a reported prevalence ranging from 9.5 to 22% ,2. Of alThere are many potential causes of neck pain, but mostly no specific underlying pathology is found so that it is designated as non-specific . AlthougIn the Netherlands, patients with neck pain are often referred for physiotherapy. Moreover, physiotherapy accounted for 84% of the total direct medical neck pain costs in 1996 [Two treatment models have been described in the literature, both of which are applicable within the field of physiotherapy. The first, a biomedical model, considers pain to be a sign of physiological damages and treatment according to this model aims to remove the pathologic condition so that the pain will no longer occur ,10. MoreMany conservative physiotherapeutic treatments are available for treating neck pain, but there is insufficient evidence to allow to conclude that one type of treatment is more effective then others ,20.In a review on chronic pain, operant behavioural therapy was found to be beneficial to waiting list control groups on outcomes such as pain experience, mood effect other than depression, social role, and for the expression of pain behaviour . CompareIn summary, it remains unclear which type of conservative, including behavioural, treatment is to be preferred in the management of chronic neck pain. Therefore, this study, Ephysion (Effectiveness physiotherapy in neck pain), aims to evaluate the clinical and cost effectiveness of an operant behavioural programme  compared with a physiotherapy treatment in patients with chronic non-specific neck pain. In addition, we aim to identify subgroups of patients who benefit most from one of the two treatments, and to identify the most important determinants for recovery from chronic non-specific neck pain.Because a biased study design can produce incorrect conclusions, the design of a trial should be carefully examined before adopting its conclusions . A desigA randomised clinical trial (RCT) has been designed to assess the effectiveness of behavioural graded activity compared with physiotherapy treatment in patients with chronic non-specific neck pain. The study design has been approved by the Medical Ethics Technical Commission of the Erasmus MC, University Medical Centre in Rotterdam and is in compliance with the Helsinki Declaration.Forty general practitioners (GP) in region West Brabant in the Netherlands will select the patients. Patients are eligible if they are aged between 18 and 70 years old, have suffered from neck pain for over three months, and have an adequate knowledge of the Dutch language. Excluded are patients diagnosed with a specific disorder , those who have had physical/manual therapy during the previous six months, those with a chronic disease (e.g. rheumatoid arthritis or coronary artery disease), or those who have to undergo surgery in the near future. Eligible patients will receive an information leaflet from their GP and the GP then informs the research department.Thereafter, the research assistant contacts the patient, provides additional information about the implications of participation, re-checks the eligibility of the patient, and completes the informed consent procedure.The sample size for this study is calculated according to the global perceived effect (GPE). Based on previous studies, a 20% difference in GPE is expected after completion of either treatment (9 weeks) and is considered to be clinically relevant; 160 patients are needed to detect this difference. In this calculation a power (1 - β) of 80% is taken into account. Thus, the inclusion of 80 patients per treatment group is planned.An independent examiner using a computer-generated randomisation schema performs randomisation. To prevent unequal distribution, patients are pre-stratified based on three important prognostic factors: gender, age and the severity of the complaint, which are recorded at baseline . FurtherPatients are told to receive physiotherapy but are blinded to allocation of the two treatments; the content of the treatments is not described in the information leaflet. This enhances the quality of the study, because the patients themselves measure the effect of treatment. GPs are also blinded for allocation to prevent accidentally informing the patients of the allocated treatment. The physiotherapists are not blinded for allocation, but the physiotherapists from each treatment group are kept strictly separate and are not involved in the outcome measurement. Finally, the primary investigator is blinded for patients' allocation but the research assistant is not; neither is involved in the outcome measurement.After receiving written information, 34 physiotherapists in region West Brabant will participate in either the physiotherapy treatment (PT) or the graded activity programme (GAP). To optimise the contrast between the two treatments, both groups are strictly separated throughout the study. The PT group consists of 16 physiotherapists and the GAP group of 18 physiotherapists. The PT physiotherapists participate in a meeting to standardize the physiotherapy treatment. The GAP physiotherapists are instructed on the behavioural graded activity approach during a two-day theoretical and practical training course.Both interventions are performed in an outpatient setting. A maximum of 18 treatments per patient is set and each treatment takes about 30 minutes, which is in accordance with medical insurance policy in the Netherlands. Before treatment starts, physiotherapists receive a completed questionnaire about the patient's main complaints ; this quThe content of the physiotherapy treatment is decided by consensus among the participating PT physiotherapists. Treatment is according to a biomedical model, which implies guidance based on the amount and severity of pain that the patient experiences.By consensus, the physiotherapy treatment is divided into the patient's primary therapy aim, three general treatment goals, and several techniques to attain those goals. The primary therapy aim is defined as the result the patient wants to achieve by the end of therapy. A general treatment goal is a goal for each single treatment and could, therefore, differ per treatment session. Table An operant approach was the basis of the behavioural graded activity programme as used in this study. The treatment is according to a biopsychosocial model, which implies that it is guided by the patients' functional abilities and that time-contingent methods are used to increase the activity level of the patient . The behBefore starting the baseline phase, the treatment vision and the patient's ideas about pain and its causes are discussed. The development and maintenance of pain will be explained and patients are reassured that it is safe to move and to increase their level of activity ,13,30. BDuring the treatment phase, patients systematically increase the time-contingent quotas to enable them to reach their personal aims within a pre-set therapy time period. To ensure a successful experience during the first exercise, the start quota is below the mean baseline score. The pre-set exercise quotas have to be strictly followed; neither over-performance nor under-performance is allowed. During this phase the patient has to practice at home and document every activity or exercise on a performance chart. These charts will be discussed in the following treatment session and achievements will be reinforced while disregarding pain behaviours. Positive reinforcements of healthy behaviour and the patient's experiences of success are considered to be important to enhance the patient's motivations.The generalization phase takes place at the end of the treatment phase. In this phase generalization of learned behaviour and management of relapses will be discussed.Baseline questionnaires are sent after inclusion, which is as soon as possible after patients have consulted their GP. Outcome of intervention will be assessed at 4 and 9 weeks after randomisation; however, if the treatment is not finished at 9 weeks, the patients will receive an additional questionnaire (Ts) after finishing the treatment. Follow-up assessments are planned at 26 and 52 weeks after randomisation.All outcome measures are reported by means of mailed questionnaires. Table Primary treatment outcome of this study is the global perceived effect, which is used to assess recovery from the complaint . In addiCosts are measured using a combination of questionnaires to collect data on direct medical costs , and indirect costs due to sick leave and disability.Secondary outcome measures include main complaints, pain intensity, medical consumption, coping, functional status, quality of life, and psychological variables. Prognostic factors are measured including demographic variables, the baseline variables and the psychological variables table .Descriptive statistics will be used to examine comparability of baseline data between PT and GAP, and to check if randomisation was successful. Before this analysis, decisions about differences considered to be clinically relevant are made and, if necessary, adjustment will be made for these differences in multivariate analysis. Further, all outcome data will be screened for normality and, if necessary, logarithmic transformations or non-parametric methods of analysis will be applied.The first aim is to evaluate the clinical and cost effectiveness of GAP compared to PT. Clinical effectiveness will be examined with a Student's t-test (continuous), a Chi-square test (dichotomised) or a Wilcoxon test  according to the intention-to-treat principle. This means that patients will be analysed in the treatment group to which they are randomly allocated. For missing data, imputation techniques will be used. When the dropout rate is 10% or more, or loss to follow-up is 20% or more, per-protocol analysis will be performed. The results on primary outcome will be dichotomised into improved versus not improved. Improved implies completely recovered and much improved, whereas not recovered implies slightly improved, not changed, slightly worsened, much worsened, and worse than ever .Cost effectiveness will be calculated from a societal perspective. Costs (direct as well as indirect) will be related to the treatment effects, based on the primary outcome measure, by calculating cost-effectiveness ratios.The second aim is to identify subgroups of patients that benefit most from one of the two treatments. The following subgroups will be investigated: duration and severity of the complaint, depression, and fear of movement.The third aim is to identify important variables for recovery. For this purpose multivariate analysis will be performed to investigate the influence of prognostic variables and patient characteristics on the outcome. Separate analyses will be conducted to investigate prognostic factors for short-term (3 months) and long-term (12 months) recovery.This study is designed to evaluate the clinical and cost effectiveness of a behavioural graded activity programme compared with a physiotherapy treatment in patients with chronic non-specific neck pain. Since physiotherapists perform both treatments in this study, contrast between the two treatments is a very important issue. There are contrasts both in the composition of the treatment and the way the physiotherapists approach the patient. With regard to the composition, the graded activity programme (GAP) starts with a systematically performed baseline measurement; this is in contrast to the physiotherapy treatment (PT), where treatment is based on history taking and physical examination. In GAP quotas are set based on the patient's behaviour, whereas in PT they are set based on pain levels and training principles. After quotas are set GAP uses a time-contingent treatment approach, which involves a pre-set systematic increase in activities. In contrast, PT uses a pain-contingent approach, which means that treatment is adapted to the patient's reaction to previous treatment sessions. Furthermore, GAP uses a hands-off approach, whereas PT may contain hands-on techniques, such as massage, traction etc Table .This study addresses an important question because chronic neck pain is a common complaint and it remains unclear which type of physiotherapeutic treatment is most effective. Recruitment of patients will take place until up to the end of 2004; follow-up measurement will continue up to end 2005.The authors declare that they have no competing interests.APV and BWK conceived the study, developed the design of the randomised clinical trial and participated in writing the article. MG is an expert in the field of graded activity and contributed to the content of the article. CJV advised on the content of the article. FV conducts the research, participated in the completion of the study design and wrote the article. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Female mate choice may be adaptive when males exhibit heritable genetic variation at loci encoding resistance to infectious disease. The Hamilton-Zuk hypothesis predicts that females should assess the genetic quality of males by monitoring traits that indicate health and vigor (condition-dependent choice, or CD). Alternatively, some females may employ a more direct method of screening and select mates based on the dissimilarity of alleles at the major histocompatibility loci (we refer to this as opposites-attract, or OA). Empirical studies suggest that both forms of mate choice exist, but little is known about the potential for natural selection to shape the two strategies in nature.We used computer simulation models to examine the evolutionary fates of the two forms of mate choice in populations at risk for infection by debilitating parasites. We found that populations exhibiting random mating (no mate choice) can be invaded and replaced completely by individuals practicing CD type mate choice. We also found that an allele encoding OA choice can increase when rare in randomly mating populations, but that it does not go to fixation under selection. A similar result was obtained when the OA strategy was introduced into populations practicing CD mate choice. As before, we found that the OA choice allele will increase when rare, and that it will not go to fixation under selection. The converse however was not true, as CD individuals gain no rare advantage when introduced into an OA population.Taken together, the results suggest that, when rare, OA is the best strategy for parasite evasion (of those considered here). The consequence of OA increasing in the population, however, is to reduce the parasite driven genotype oscillations and facilitate the breakdown of linkage disequilibrium at the disease-resistance loci. This leads to a neutrally stable situation in which different strategies have equal fitness, and suggests that multiple forms of mate choice may be expected to occur in populations at risk from infectious disease. Since Darwin published his classic work on sexual selection , biologiIn a previous study, we investigated the potential for active mate choice to indirectly favor the maintenance of sexual reproduction . We founThe results from our study show that when populations are confronted with infectious disease, alleles encoding OA and CD mate choice both increase when rare in randomly mating populations Figs.  and 2A. As the allele for OA increased in the population, the oscillations in both host and parasite resistance genotypes underwent a dramatic reduction in amplitude Fig . In addiIn contrast, as the allele for CD choice increased from its rare starting point, the oscillations in genotype frequency increased, rather than decreased Fig. . This reUnder the conditions studied here, both OA and CD alleles increase when rare in randomly mating populations, but only the CD allele goes to fixation due to sexual selection. How does CD fare against an OA population? We found that when introduced into a population of 20,000 OA individuals, a rare allele encoding CD choice was not favored by selection, but that it could become established and increase in frequency as a result of genetic drift Fig. . This reIn contrast, OA did increase when rare due to selection in a population of CD individuals; hence CD is not an ESS Fig. . The oveAmong the three strategies of mate choice for parasite avoidance considered in this study, only OA is evolutionary stable. However, OA will not eliminate either RM or CD-choice through the action of sexual selection, since OA becomes selectively neutral at intermediate frequencies in both situations by causing reductions in the fitness variance among resistance genotypes. Thus, OA and RM, OA and CD, or all three could coexist in the short term in large populations (until OA goes to fixation by drift). As such, in populations at risk from disease, females might practice more than a single type of mate choice.The methods employed in this investigation are similar to those used in our previous studies of host parasite coevolution ,25. We uT. For the two locus version of the model, T was set to a value of 0.8, and parasites underwent two generations for each host generation. In the three-locus, five-allele version of the model, higher rates of parasite transmission coupled with a greater asymmetry of host and parasite generation times were required to sustain infection in the host population. For this version of the model, T was set to a Poisson distributed mean of eight, and parasites underwent ten generations for each host generation. In all cases, if a parasite matched a host exactly at all interaction loci, the host was marked as infected and the parasite was placed in a pool of potential reproductives. Once infected, individual hosts were protected against further attack by parasites. Reproduction in hosts and parasites was accomplished by drawing individuals from their respective populations at random with replacement. The parasite life cycle included a "free-living" stage in which reproductive adults emerged from infected hosts to mate. When an individual (host or parasite) was selected for reproduction, a second individual was randomly selected for cross-fertilization. Gametes from the two haploid "parents" were then brought together to form a diploid zygote stage, where free recombination between the interaction loci made possible the production of a genetically diverse brood of haploid offspring. All else equal, sexual individuals (hosts and parasites) produced a lifetime average of 10 haploid offspring. The number of offspring produced by hosts, however, was reduced according to the status of parasitic infection for each of the parents. The reproductive output of each host parent was discounted according to 10(1-E), where E simulates the detrimental effect of parasitism on host reproduction (virulence). In the present study, E was set to a value of 0.8 to simulate the effect of a moderately virulent parasite. Following reproduction, a maximum of 20,000 parasite and 20,000 host offspring were selected at random to become the next generation of adults.During each parasite generation, hosts were drawn sequentially and exposed to a randomly drawn parasite with a probability of n, where n was the total number of alleles present at each locus. Prior to data collection, the simulation was allowed to run for 200 host generations; this allowed for establishment of long-term coevolutionary dynamics arising from the host-parasite interaction. Next, we tracked the fates of alleles encoding different types of mate choice in the host population. Mating preferences in hosts were controlled by a single locus encoding one of three strategies: random mating (RM), condition-dependent mate choice (CD), or opposites attract (OA) mate choice. Random mating was simulated by pairing two randomly drawn adults. Condition-dependent choice was implemented by allowing the first reproductive drawn  to identify and discriminate against parasitized "males". For the opposites attract strategy, females preferred "males" with alleles different from their own at each of the two interaction loci. For both OA and CD choice, "females" were allowed to choose the "best" available male from a sequence of 20 randomly drawn "males".At the beginning of each run, the interaction alleles at each locus in the host and parasite populations were initialized to a frequency of 1/RSH wrote the computer code and implemented the simulations. Both authors participated equally in all other phases of the work.


Is evolutionary theory is incomplete and are we failing to understand phenomena as disparate as ecosystem development and the interplay of genes and culture in shaping human evolution? This book, the latest in the excellent Monographs in Population Biology series from Princeton University Press, is a work of advocacy in which the authors argue that evolutionary theory is incomplete and that, in consequence, we are failing fully to understand phenomena as disparate as ecosystem development and the interplay of genes and culture in shaping human evolution. What we are missing, they argue, is an appreciation of niche construction, the process by which an organism modifies the abiotic and biotic environment in which it is subject to natural selection. The authors' major assertion is that the importance of niche construction is so great that it should be regarded “after natural selection, as a second major participant in evolution” and that it is “not just an important addition to evolutionary theory” but “requires a reformulation of evolutionary theory”. Bold claims indeed.After introducing the conceptual framework Odling-Smee et al. set out a series of arguments to support this position, the first of these being the empirical case for the existence of niche construction. Niche construction, as broadly interpreted here, is everywhere. Animals build nests, burrows, and protective cases and so alter the environment they experience in a way that may select for further adaptations. The changes caused by some animal species, such as beavers and earthworms, are of a sufficient magnitude that the environment experienced by a host of other species is affected. Many plant species also modify the environment they experience by generating organic litter; influencing hydrological and biogeochemical cycles; affecting temperature, humidity, and light regimes; and, over the longer term, determining the make up of the atmosphere. Decomposer and chemoautotrophic microorganisms similarly influence biogeochemical transformations, while parasitic species can manipulate the behaviour and internal environment of the hosts they infect. Perhaps less obvious examples of niche construction are the many types of migration and cultural evolution that, like physical transformations, cause the organism's descendants to experience a different selective environment.After this broad, accessible survey, the authors change key rather abruptly and explore two-locus, frequency-dependent population genetics. The novelty here is that selection on one locus depends on the history of gene frequencies at the other, “niche construction”, locus. In an extension, gene frequencies at one locus affect an environmental variable with its own dynamics that in turn influences the second locus. As one would guess, the models display a range of potentially interesting dynamics, though generalisations and broad conclusions are sparse. We guess the aim of the chapter is to illustrate that environmental feedbacks can be potent and general agents of evolutionary change, but the restriction of the theory to such a narrow model, with very technical explanation, risks losing the few readers who we suspect will stay the course .Perhaps aware of the dangers of getting bogged down in detail, the argument then moves to proving a case for the universality of niche construction. Invoking the second law of thermodynamics and Maxwell's Demon, the authors lead us through a challenging thesis that concludes that the persistence of life on earth requires both natural selection and niche construction, thereby justifying some of the bold claims for their new theory. We think they are technically correct, but we are concerned that the demonstration of the inescapability of niche construction, as defined here, does not guarantee that it will actually tell us new and important things about the world, as the theory of natural selection has.The remainder of the book explores the implications of niche-construction thinking for evolutionary biology, ecology, and the human sciences, and in our view is the most successful part. Though it is rare for the authors to offer new analysis and insight, their sideways look at many issues from the niche-construction viewpoint often offers interesting new angles on old problems, and suggests new avenues of enquiry that may be the book's greatest legacy. A good example of this is their convincing and timely argument that a more explicit recognition of evolution's role in environmental feedbacks will help to unify population/community ecology and ecosystem science.avant la lettre, has given way to a more pluralistic theory of succession. It is a great pity that the authors give so little space to plants and plant ecology, as it here that some of the finest examples of niche construction are found, as well as the best-developed conceptual framework for studying the roles of environmental feedback.The chief argument for the prosecution is that niche construction is common but not pervasive, and that wherever ecologists and evolutionists have found interesting examples of it, they have developed appropriate theory and concepts to understand its ramifications. For example, some of the clearest examples of niche construction occur in plant succession where, as F.E. Clements realised nearly a hundred years ago, early-succession plants frequently modify their environment in ways that allow other species to replace the pioneers. Interestingly, the strict Clementsian theory of facilitation, niche construction Other areas where biologists have well-developed theories of the influences and impacts of niche construction include co-evolutionary theory, where the environmental feedbacks are largely biotic, and Dawkins' theory of the extended phenotype. Very close to some of the arguments discussed here, and generously acknowledged, is the idea developed by Jones, Lawton, and colleagues of ecosystem engineers, species that have a major impact on the abiotic environment experienced by a large number of species.A major strength of the book is that it reveals common processes and patterns underlying disparate biology in consistently interesting ways. Its chief contribution is thus not to tell us new things about how nature works but to link together many different aspects of ecology under an umbrella of theory that may in the future lead to new insights. Do they deliver on their grand claims? Time will tell, but our view is that they don't. They engagingly admit that for their project to succeed the new theory must earn its keep by producing significant new biology—something which has yet to occur. However, the great need for the biological and human sciences to integrate across subdisciplines, as the authors bravely attempt here, makes this a hugely worthwhile book. Its breadth of scope and its boldness in creating syntheses have resulted in a stimulating and challenging read.


Pao/BEL family is probably the least well studied. It is becoming apparent that numerous LTR retrotransposons and other mobile genetic elements have colonized the genome of the human blood fluke, Schistosoma mansoni.Of the major families of long terminal repeat (LTR) retrotransposons, the Sinbad, a new LTR retrotransposon, was identified in the genome of S. mansoni. Phylogenetic analysis indicated that Sinbad belongs to one of five discreet subfamilies of Pao/BEL like elements. BLAST searches of whole genomes and EST databases indicated that members of this clade occurred in species of the Insecta, Nematoda, Echinodermata and Chordata, as well as Platyhelminthes, but were absent from all plants, fungi and lower eukaryotes examined. Among the deuterostomes examined, only aquatic species harbored these types of elements. All four species of nematode examined were positive for Sinbad sequences, although among insect and vertebrate genomes, some were positive and some negative. The full length, consensus Sinbad retrotransposon was 6,287 bp long and was flanked at its 5'- and 3'-ends by identical LTRs of 386 bp. Sinbad displayed a triple Cys-His RNA binding motif characteristic of Gag of Pao/BEL-like elements, followed by the enzymatic domains of protease, reverse transcriptase (RT), RNAseH, and integrase, in that order. A phylogenetic tree of deduced RT sequences from 26 elements revealed that Sinbad was most closely related to an unnamed element from the zebrafish Danio rerio and to Saci-1, also from S. mansoni. It was also closely related to Pao from Bombyx mori and to Ninja of Drosophila simulans. Sinbad was only distantly related to the other schistosome LTR retrotransposons Boudicca, Gulliver, Saci-2, Saci-3, and Fugitive, which are gypsy-like. Southern hybridization and bioinformatics analyses indicated that there were about 50 copies of Sinbad in the S. mansoni genome. The presence of ESTs representing transcripts of Sinbad in numerous developmental stages of S. mansoni along with the identical 5'- and 3'-LTR sequences suggests that Sinbad is an active retrotransposon.A proviral form of Sinbad is a Pao/BEL type retrotransposon from the genome of S. mansoni. The Pao/BEL group appears to be comprised of at least five discrete subfamilies, which tend to cluster with host species phylogeny. Pao/BEL type elements appear to have colonized only the genomes of the Animalia. The distribution of these elements in the Ecdysozoa, Deuterostomia, and Lophotrochozoa is discontinuous, suggesting horizontal transmission and/or efficient elimination of Pao-like mobile genetic elements from some genomes. Schistosoma mansoni, the African blood fluke and etiological agent of intestinal schistosomiasis, is endemic in numerous countries in Africa, the Middle East, the Caribbean and northeastern South America. The life cycle of S. mansoni involves parasitism of both humans and aquatic snails of the genus Biomphalaria. Cercariae, the infectious larvae, emerge from the snails into lakes and fresh water streams, where they initiate human infection by direct penetration of the skin. Within the infected person, the worms develop into male and female adults within the portal system blood vessels and mesenteric veins of the intestines. Eggs released from the female parasite into the blood traverse the intestinal wall and are passed out with the feces. Among the tropical diseases, schistosomiasis ranks second only to malaria in terms of morbidity and mortality [ortality  and has S. mansoni are of interest both for their potential in developing tools for schistosome transgenesis and for their influence on the evolution and structure of the schistosome genome [Boudicca, the first LTR retrotransposon characterized from the genome of S. mansoni [gypsy -like retrotransposons, one of three highly divergent groups of LTR retrotransposons: the Gypsy/Ty3 group, the Copia/Ty1 group and the Pao/BEL group [Mobile genetic elements (MGEs) represent a major force driving the evolution of eukaryotic genomes -4 and ple genome ,14. Preve genome ,16, longe genome ,17,18, ne genome ,11, and e genome . Boudicc mansoni  belongs EL group . AlthougEL group ,11,22, aEL group , suggestEL group  and DNA EL group ,26. FurtEL group ,20.Pao-like elements exhibit a host range limited to insects and nematodes [Pao-like sequences have been reported from vertebrates including the teleost fishes Takifugu rubripes and Danio rerio [Pao-like element from the genome of S. mansoni, which we have named Sinbad after the mariner-explorer Sinbad from the classical Persian/Arabic tales of the "1001 Arabian Nights" . (Sinba" (e.g., .) FurthePao and other Pao-like retrotransposons including Ninja and MAX (not shown). Using a probe based on an RT encoding segment of the end sequence of BAC 30-H-16, we identified 14 positive clones in the S. mansoni BAC library [Pao and Ninja, followed by other Pao/BEL type retrotransposons. We have termed this new retrotransposon Sinbad. The coding region between the two LTRs of Sinbad was disrupted by several stop and frameshift mutations -encoding sequence with identity to  library . DotPlot library . BLAST ssee Ref. ), althouSinbad revealed the presence of an ORF encoding retroviral gag and pol-like proteins. A multiple sequence alignment of some of the key structural and enzymatic domains is presented in Figure Sinbad sequence and orthologous regions from Pao, Roo, BEL, MAX and Ninja. The Cys-His box is a highly conserved cysteine and histidine based motif of the nucleocapsid protein (part of the gag polyprotein) of retroviruses and retroviral like elements [Pao-like elements are characterized by a distinctive triple Cys-His box [CX2CX3-4HX4C, CX2CX2-4HX4-5C, and CX2-4CX3HX4H. Sinbad also exhibits the latter type, hallmark triple Cys-His box motif  domain of ~260 amino acids in length. Integrase mediates integration of a DNA copy of the viral genome into the host chromosome. Integrase is composed of three domains, the amino-terminal zinc binding domain, a central catalytic domain, and a carboxyl terminal domain that is a non-specific DNA binding domain [BEL/Pao-like retrotransposons including MAX, Saci-1, Pao, Ninja, Roo, Suzu, BEL, and Tas as well as Sinbad is presented in Figure Sinbad sequence. The NH2-terminal zinc-finger region of Sinbad included two conserved Cys residues and one His residue characteristic of other zinc finger motifs of IN D(49)E. The IN of non-Pao/BEL retrotransposable elements, for example, Copia, exhibit a DD(35)E motif [BEL/Pao like elements is unusual in that there is an expanded number of residues between the second D and E conserved residues, with DD(45)E for Pao and DD(53)E for BEL. Sinbad conformed to this BEL/Pao-like paradigm with a spacing of DD(49)E. Saci-1, also from S. mansoni, shows DD(49)E, although the IN domain of these two elements exhibited only 52% identity. The carboxy terminal domain of IN of Sinbad extended about 135 amino acids beyond the E residue of the catalytic domain [see An RNaseH domain spanning ~300 amino acid residues was located carboxyl to RT, in which the conserved active site motif DAS was apparent see . At its g domain . A multiN Figure . A secon)E motif . Howevermain see .Sinbad exhibited identity to Saci-1 from S. mansoni, and indeed these Pao-like retrotransposons from S. mansoni share substantial identity in deduced amino acid sequence and in structural organization [Sinbad and Saci-1, they also demonstrated that Sinbad and Saci-1 are distinct retrotransposons. Finally, Sinbad did not appear to encode an envelope protein, the retroviral gene product necessary for extracellular existence and infection [As noted, the IN of nization . This sinfection .Sinbad was aligned with that of 19 Pao/BEL retrotransposon family elements, and with RT from informative Gypsy-like elements, from HIV-1, and Copia using ClustalW. Bootstrapped trees were then assembled using the neighbor joining method and Njplot. Copia was employed as the outgroup to root the tree. The phylogenetic tree confirmed that Sinbad belonged to the Pao/BEL family of LTR retrotransposons . Sinbad also grouped closely with Pao and Ninja. Sinbad is clearly distinct from the Gypsy-like retrotransposons, including Gulliver of Schistosoma japonicum and Boudicca of S.mansoni. Sinbad is also clearly distinct from HIV-1, representative of vertebrate retroviruses, and from Copia, representative of the Ty1/Copia group of LTR retrotransposons. Among the 20 BEL/Pao family elements represented in the tree, it was possible to distinguish several subfamilies. First, the outlying subfamily was a clade including Suzu (from T. rubripes) and an unnamed element from zebrafish. These are the only two elements that we have observed in this subfamily, and both occur in fish genomes. The other two branches of these retrotransposons include Pao, on the one hand, and BEL on the other. Moreover, two subfamilies of elements were apparent within each of the Pao and BEL branches. For the Pao branch, one sub-family included Pao (from B. mori), ninja (from D. simulans) and an unnamed element from Anopheles gambiae (XP_3092181). These subfamily elements were all from insect genomes. The other subfamily included Sinbad, Saci-1 and the D. rerio element BK005570; this subfamily has elements from schistosomes (Phylum Platyhelminthes) and fish. On the BEL branch of the tree, the first subfamily includes elements solely from nematode genomes – Tas (A. lumbricoides), several Cer elements from C. elegans, and an unnamed element from C. briggsae (BK005572). The other branch included BEL itself (from D. melanogaster), Kamikaze (B. mori), MAX (D. melanogaster) and Moose from A. gambiae. Members of this fifth subfamily occurred only in insect genomes.The RT domain of s Figure , and revPao/BEL family retrotransposons. The tree displayed the same general topography of branches as the RT-based phylogram and supported our suggestion that there are (at least) five discrete sub-families of BEL-Pao family retrotransposons: Tas-like, BEL-like, Pao-like, Sinbad/Saci-1-like, and Suzu-like . In similar fashion to the RT based tree, Sinbad and Saci-1 were closely related to each other and to the IN from the unnamed Pao-element from zebrafish (BK005571).In addition, a phylogram of IN sequences was assembled from 14 S. mansoni gDNA, S. japonicum gDNA and BAC 33-N-3 confirmed the presence of Sinbad in the S. mansoni genome but indicated it was absent from the genome of the related schistosome, S. japonicum  were strong and dispersed, with a band of ~2.6 kb in the Hind III digest. The smeared pattern of hybridization indicated that a number of copies of Sinbad were interspersed throughout the genome of S. mansoni rather than being localized at a discrete locus. By contrast, the probe did not hybridize to the gDNA of S. japonicum. Additional blots with larger amounts (30 μg) of S. japonicum gDNA, digestion with BamH I instead of Hind III, and exposure of the film for longer periods failed to yield any signal from S. japonicum gDNA (not shown), indicating that Sinbad was absent from this schistosome species. Strong hybridization signals were evident in the positive control lanes of digests of BAC 33-N-3. Densitometric analysis of the hybridization signals indicated the presence of 50 to 60 copies of Sinbad per S. mansoni haploid genome, based on four separate estimates comparing the signal in each of the genomic DNA lanes to the signal in each of the 33-N-3 BAC lanes (comparison of lane 1 with lane 4, comparison of lane 2 with lane 5, comparison of lane 1 with lane 5, and comparison of lane 2 with lane 4). (These estimates assumed that BAC 33-N-3 included only one copy of Sinbad.)Southern hybridization analysis of m Figure . BamH I S. mansoni genome, approximately 0.7% to 1.0% of the clones were positive, indicating a copy number for Sinbad of ~20 to 30 copies (not shown). Second, the bioinformatics approach of Copeland et al. [Sinbad (Table Hind III [Hind III sites will be underrepresented in the BAC end sequences. Accordingly, since sequenced BAC ends from this library constitute a large proportion of the genomic S. mansoni sequences in the public domain, we used only genes containing Hind III sites as reference sequences. As shown in Table Sinbad, 38, was higher than the number of hits for the single-copy cathepsin D gene (0 hits) but lower than that for the multiple-copy 28S ribosomal RNA gene (157 hits) , SR2 (102 hits, 1,000–10,000 copies), and SR1 (104 hits, 200–2,000 reported copies). In overview, all three methods were in reasonably close agreement, and together they indicated that approximately 50 (range ~20–100) copies of Sinbad reside in the genome of S. mansoni. Based on copy numbers estimated for other schistosome retrotransposons , we conSinbad as the query sequence and the GenBank EST database of non-human, non-mouse sequences. The database includes more than 130,000 EST sequences from six developmental stages of S. mansoni – egg, miracidium, cercaria, germball (= sporocyst), schistosomulum, and mixed sex adults [Sinbad, CD111741, CD060185, CD163413, CD062550, CD156994, and CD156946, exhibited contiguous ORFs spanning each EST without frameshifts or stop mutations. Positive ESTs spanning most or all of the LTR, gag, PR, RT, RH and/or IN regions were located in most of these six developmental stages, indicating that Sinbad-like elements are actively transcribed in all or most developmental stages of S. mansoni.BLASTn analyses were undertaken using the full length of x adults ,40. SignSinbad-like retrotransposons, we examined numerous complete and partial genomes, including prokaryotes, plants, fungi, animals, and lower eukaryotes [Sinbad spanning from the Cys-His box to the conserved protease catalytic domain (bp 1588–2236) [see Sinbad-like elements and members of the Pao/BEL family at large. No significant hits were found in any of the plant, fungal, or protist genomes examined, or in the 275 bacterial and 21 archaean genomes searched. All of the nematodes examined were positive. Of the insects, the other branch of the Ecdysozoa, Drosophila melanogaster and Anopheles gambiae contained Sinbad-like elements, whereas Drosophila pseudoobscura and Apis mellifera did not. Of the vertebrates, Danio rerio and Takifugu rubripes contained Sinbad-like sequences, whereas Homo sapiens, Mus musculus, Rattus norvegicus, Canis familiaris, Sus scrofa, Gallus gallus, and Bos taurus did not. Interestingly, although most higher chordates examined were free of Sinbad-like elements, the tunicates Ciona intestinalis and Ciona savigny, were positive for over 100 hits of sequences highly similar to the Sinbad search sequence . In addition, the echinoderm Strongylocentrotus purpuratus, a non-chordate deuterostome, was positive for the Sinbad search sequence. These findings are summarized in a tree-of-life style illustration, based on the tree presented in Pennisi [Sinbad are marked with a "+" symbol and those without are indicated with a "-" symbol. The results of a search of dbEST corroborated and expanded these findings, revealing nine non-Schistosoma organisms with Sinbad -like sequences: C. intestinalis, Molgula tectiformis (tunicate), S. purpuratus, D. melanogaster, Bombyx mori, Salmo salar, Xenopus laevis, and Trichinella spiralis. E-values and accession numbers for the top match for each organism are provided in Table In order to examine the phylogenetic distribution of karyotes . The gen236) see  as the q Pennisi , and dra Pennisi . E spacing of the active site aspartic acid and glutamic acid residues. The YVDD motif of RT, a version of the F/YXDD consensus motif of Gypsy-like LTR retrotransposons, is shared by Pao and BEL. Bowen and McDonald [Cer7-Cer12 series of elements from C. elegans displayed YVDN at this site. Whether the Asn could replace Asp as the carboxy-residue of this conserved tetrapeptide with retention of enzyme activity remains to be determined by biochemical analysis, although mutation of either aspartate in YXDD of retroviral RT (HIV-1 or Moloney murine leukemia virus) inactivates the polymerase [see [Although several LTR retrotransposons have been characterized previously from the genome of fugitive ,20,37, tMcDonald  reportedase [see ].Sinbad in BAC 33-N-3 are identical in sequence, and appeared to contain a putative promoter for initiation of transcription by RNA polymerase II. Along with conservation of most residues contributing to the active sites of the retrotransposon enzyme domains, these structural characteristics suggested that Sinbad is active or had been transpositionally active in the recent past. Several other features also indicated that Sinbad is transpositionally active. Numerous transcripts spanning enzymatic domains and LTRs of Sinbad, from at least six developmental stages of S. mansoni, have been sequenced [Sinbad are composed entirely of contiguous open reading frames, suggesting non-mutated copies. On the other hand, potentially inactivating mutations, including stop codons and frameshifts, suggested that the BAC 33-N-3 copy of Sinbad was incapable of autonomous retrotransposition. If active copies are present, functional proteins coded by these copies could have been used in the recent past to mobilize the 33-N-3 Sinbad copy in trans, as recorded for other retrotransposons [BEL like elements in C. elegans, implying recent transposition.The LTRs of equenced , and of nsposons -47, explnsposons  noted thSinbad, at 386 bp in length, were substantially shorter than those of Saci-1, ~840 bp [Gypsy-like LTR retrotransposons from schistosomes, the fugitive, Gulliver and Boudicca. Whereas Sinbad and Saci-1 are clearly closely related, dissimilar LTRs and the low amino acid identity of the most highly conserved domains (35 to 52%) confirmed they are distinct retrotransposons. Sinbad can be added to the catalog of mobile genetic elements characterized from the schistosome genome, where retrotransposons appear to have proliferated and flourished and contributed significantly to its relatively large size  [S. mansoni by Sinbad and Saci-1 and that of S. japonicum by the related Tiao element [Pao/BEL family LTR retrotransposons. The presence of Sinbad, Saci-1, and Tiao in two species of Schistosoma suggests that an ancestral schistosome was already host to the ancestors of these elements. The LTRs of  ~840 bp , but long genes) ,20,40. T element  represenPao-like elements, Pao from the silk moth B. mori, Tas from the human roundworm Ascaris lumbricoides and BEL from D. melanogaster have been known for about a decade, the Pao/BEL family is not as well understood or apparently as widespread as the other two major families of LTR retrotransposons, the Copia/Ty1 and the Gypsy/Ty3 families. However, at least three branches of the Pao/BEL family have become apparent – branches represented by Pao, BEL, and Suzu (from T. rubripes) [Sinbad, and some related elements, we have been able to investigate the intra-family relations of the Pao/BEL elements more thoroughly. Our findings, based on phylogeny of RT, and supplemented by phylogeny of IN, indicated the presence of at least five sub-families of Pao/BEL elements. The majority of the sub-families may have a restricted host range; the Tas subfamily occurred only in nematodes (these elements may be endogenous retroviruses because they appear to include env genes), the BEL subfamily only in insects, the Pao subfamily only in insects, and the Suzu subfamily only in fishes. By contrast, the Sinbad/Saci-1 subfamily is known from schistosomes and zebrafish.Whereas the sequence and deduced structure of the three signature ubripes) ,32,49-51Pao/BEL retrotransposons are known only from animals, a less extensive distribution than those of the Copia/Ty1 or Gypsy/Ty3 groups that include elements known from fungi and/or plants as well as animals. The ostensible absence of these elements from prokaryotes, lower eukaryotes, fungi and plants suggests that ancestral Pao-like elements appeared after the differentiation of the Animalia. Though the number of sequenced entire genomes of animals is small, the distribution of Pao/BEL LTR retrotransposons within these few genomes displays a topography that we would not expect to be the result solely of vertical transmission alone  and S. mansoni (a platyhelminth). Further, the distribution among chordates is enigmatic. Of the vertebrate whole genomes searched, only two, T. rubripes and D. rerio, were positive for Sinbad like elements. The human, mouse, rat, cow, chicken, pig and dog genomes were devoid of Sinbad-like matches. Since the genomes of lower chordates and a non-chordate deuterostome were positive for Sinbad-like sequences, progressive radiation would be expected to give rise to similar sequences in these vertebrates.The one Fig. . Sinbad-Merlin DNA transposons; Merlin like elements are abundant, for example, in anopheline mosquitoes but are absent from D. melanogaster, D. pseudoobscura, and A. mellifera. Also, they are present in some vertebrate genomes but not others. Merlin-like elements are also present in schistosome chromosomes. This type of distribution suggests that either the vertical lineage of the elements has been curtailed by the extinction of these elements from several genomes, or that horizontal transmission has taken place. Genomes need to restrain the uncontrolled proliferation of mobile genetic elements, especially retrotransposons, and indeed some eliminate mobile sequences more efficiently than others [Ngaro elements have been lost from certain genomes, as evidenced by the presence of small, corrupt fragments serving as fossil sequences. Similarly, especially in view of the low number of Sinbad copies, Pao-like elements may have followed a course of progressive radiation followed by elimination from the Sinbad-negative genomes. However, if this were the case with Pao-like elements, relic sequences could be expected in at least some of the Sinbad-negative genomes. Their absence from mammalian and avian genomes favors the alternative explanation, that the current range reflects horizontal transmission.Feschotte  reportedn others ,52. Goodn others  have shoPao/BEL radiation within the Animalia? Felder et al. [Tas and Pao may have undergone a horizontal transmission event between the Insecta and Nematoda, followed by the eventual differentiation of these elements, including the gain or loss of env. Of the sub-families of Pao/BEL elements apparent in the RT-based phylogram . The acquisition of an envelope protein by an ancestral Tas or Tas-like element would have enabled its extracellular existence and facilitated its horizontal transmission and infection of other hosts [What might have been the origin of the r et al.  suggestem Figure , the Taser hosts .Sinbad-like sequences included a sea urchin, tunicates, pufferfish, zebrafish, the Atlantic salmon, and the African clawed frog X. laevis  stages of S. mansoni which enter and exit the snail. It will be of interest to determine whether or not Pao-like elements are present in this snail host, from which numerous RT-encoding sequences already have been reported [X. laevis and S. mansoni contain Pao-like elements and that X. laevis is the secondary host of the trematode parasite Tylodelphys xenopi [T. xenopi and another human schistosome, Schistosoma haematobium, use snails of the genus Bulinus as intermediate hosts. An aquatic lifestyle is an obvious relationship that links all of the deuterostome hosts of Sinbad-like elements. This aquatic, in comparison to a terrestrial, existence may have facilitated transmission of infectious particles of the Tas-like ancestors of Pao, Tas, BEL, Suzu, Sinbad, and relatives. Alternatively, schistosomes may have acquired a Tas- element directly from Ascaris lumbricoides, an exceedingly common human parasite and the host of Tas. A. lumbricoides occurs in the intestines of infected people, as do schistosome eggs, so direct transmission of a mobile genetic element from roundworm to schistosome could have been facilitated by their physical proximity within the human intestines.Interestingly, the deuterostomes bearing s Figure . These areported . Also ofs xenopi , a flukePao/BEL like LTR retrotransposon named Sinbad is interspersed within the genome of the blood fluke, S. mansoni. About 50 copies of this element appear to reside in the S. mansoni genome. Analyses of the phylogenetic distribution of Pao/BEL-like retrotransposons indicated that Pao/BEL-like elements are present only within phyla of the Animalia, and not in prokaryotes, fungi or plants. Further, the analyses indicated that there are at least five discrete sub-families of the Pao/BEL clade of LTR retrotransposons, and that the distribution of these retrotransposons among the Ecdysozoa, Lophotrochozoa and deuterosomes has been influenced by horizontal as well as vertical transmission.A Schistosoma mansoni genome. The library, constructed in the plasmid vector pBeloBac11 with genomic DNA (gDNA) from cercariae of a Puerto Rican strain of S. mansoni partially digested with Hind III, consists of 23,808 clones, about 21,000 of which are estimated to contain inserts ranging from 120 to 170 kb, providing ~8-fold coverage of the schistosome genome. Numerous BAC end sequences determined from randomly selected clones from this library are in the public domain. Inspection of the end sequence of BAC clone number 30-H-16 indicated identity with Pao-like LTR retrotransposons (not shown). Because the retrotransposon sequence was located at the end of the BAC, the clone was unlikely to contain the entire Pao-like element. Given that retrotransposons can be expected to be present in multiple copies in the host genome, we screened the library with a probe based on the end of BAC 30-H-16 in order to locate an entire copy of the retrotransposon. The gene probe was obtained by PCR amplification of a fragment of BAC 30-H-16 using the primers 5'-CGCGGATCCAAGAGAAAAACCTTGATAGAC and 5'-CCGGAATTCCTGTCGAAGATAAAAGAGC, was cloned into pBluescript and its identity confirmed by sequencing (Accession AY871176). This probe spanned residues 2457 to 2823 of the BAC 33-N-3 copy of the new retrotransposon (see below). The cloned insert was labeled with digoxygenin (DIG) and employed to screen the BAC library, as described [BamH1 fragments of the BAC into pNEB 193 (New England Biolabs, MA), sequencing the inserts of the sub-clones, and also by direct sequencing of BAC 33-N-3. Automated nucleotide sequencing, using ABI BigDye Terminator chemistry (ABI, Foster City, CA) and an ABI Prism 3100 sequencer, was undertaken using primers specific for the probe and subsequently with gene specific primers at Tulane University and at Davis Sequencing (Davis, CA).Le Paslier et al.  describeescribed  and the escribed  to the Dgag, protease, and reverse transcriptase: Ninja, T31674; Pao, S33901; MAX, CAD32253; Roo, AAN87269; BEL, AAB03640; and Saci-1, BK004068. Sequences of the following retrotransposons were used in the multiple sequence alignment based on Integrase: Saci-1, DAA04498;Pao, S33901; Ninja, T31674; Roo, AAN87269; Suzu, AF537216, BEL, AAB03640, Tas, Z29712, and MAX, CAD32253.Contigs of the sequences were assembled using SeqMan (DNAstar, Inc., Madison, WI). Repeat sequences were identified with a Pustell style dot matrix  using thS. mansoni and of adults of a Chinese (Anhui Province) strain of S. japonicum were extracted using the AquaPure Genomic DNA Purification system (Bio-Rad, Hercules, CA). S. mansoni gDNA (30 μg/lane) and 33-N-3 BAC DNA (800 ng) were digested with Hind III and BamH I restriction enzymes, and S. japonicum gDNA (20 μg/lane) was digested with Hind III. Digested gDNA and BAC DNA were size fractionated by electrophoresis through a 0.8% agarose gel, transferred to a nylon membrane (Zeta-Probe GT, Bio-Rad) by capillary action [Sinbad, according to the formula [(A/B) × C]/E = F. This formula was derived from two equations: (A/B) × C = D and D/E = F, where A was the number of copies of Sinbad in the BAC 33-N-3 lane, B was the density volume of the 33-N-3 lane in units of optical density per mm2, C was the density volume of the S. mansoni genomic DNA lanes in units of optical density per mm2, D was the total number of copies of Sinbad per genomic DNA lane, E is the number of haploid genomes in the gDNA lane, and F represented the copy number of Sinbad per haploid S. mansoni genome. The insert of 33-N-3 was estimated to be 145 kb in length and assumed to contain only a single copy of the retrotransposon.Genomic DNAs of cercariae of a Puerto Rican strain of y action , and UV-cum gDNA  μg/lane Sinbad retrotransposon also were obtained by a comparative bioinformatics approach [S. mansoni genomic sequences targeted more well-characterized retrotransposable elements from S. mansoni for which copy numbers had been reported. These included the Boudicca LTR retrotransposon [SR1 and SR2 [S. mansoni, all of which included at least one Hind III site. Specifically, the Advanced BLAST function was used, set to search only the S. mansoni sequences in the GSS database (Limit by Entrez Query: <Schistosoma mansoni[organism]>), and with the E value at 0.000001. The E value  reflects the probability of obtaining a match purely by chance. Scores at or below this stringent cutoff E value of 10-6 were counted as positive. This exceptionally stringent cutoff value was used to minimize the chance of counting other Pao-like elements in the total copy number of Sinbad. Since the formula for E is based not only on the bit scores of the local alignment of each pair of sequences, but also on the lengths of the subject and query [see [In addition to the densitometry-based estimate, estimates of the copy number of the approach  wherein ansposon  and the  and SR2 ,62. The ery [see ], no addTy3, S53577; Tas: Z29712; Suzu, AF537216; Sinbad, AY506538 (an N was inserted at position 2761 to a resolve a frameshift and generate a single ORF) Saci-1, DAA04498; Roo, AAN87269; Ninja, T31674; Moose, AF060859; Max, CAD32253; Kamikaze, AB042120; HIV-1, P04585; Gypsy, GNFFG1; Gulliver, AF243513; Copia, OFFCP; BEL, AAB03640; Cer7, AAB63932, Cer8, CAB04994, Cer9, CAB1647, and Cer11, AAA82437, two uncharacterized Anopheles gambiae retrotransposons, XP_309281 and XM_308737, an uncharacterized Caenorhabditis briggsae retrotransposon, AC084491, and two uncharacterized Danio rerio retrotransposons, BX537152 and BX005079 [see Pao was a reconstruction prepared by Abe et al. [Boudicca was a composite of translated cDNA sequences introduced in Copeland et al. [Sequences for phylogenetic analysis comparing the RT region of several different retrotransposons were prepared by trimming sequences from the large single polyprotein of each retrotransposon to just the conserved domains of RT see ,27). Pol. Pol27])5079 see . Two addd et al. .Sinbad. The deduced amino acid sequence encoding the region from the Cys-His Box through to the protease domain (encoded by nucleotides 106 to 753 of Sinbad [Y506538]) was employed as the query to search each genome individually using tBLASTn. The genomes searched in this way were as follows: Homo sapiens, Mus musculus, Rattus norvegicus, Takifugu rubripes, Danio rerio, Bos taurus, Gallus gallus, Sus scrofa, Canis familiaris, Anopheles gambiae, Apis mellifera, Drosophila melanogaster, Drosophila pseudoobscura, Brugia malayi, Caenorhabditis elegans, Caenorhabditis briggsae, Strongylocentrotus purpuratus, Ciona intestinalis, Ciona savigny, Giardia lamblia, Plasmodium falciparum, Plasmodium yoelii, Plasmodium berghei, Cryptosporidium parvum, Eimeria tenella, Theileria annulata, Toxoplasma gondii, Dictyostelium discoideum, Entamoeba histolytica, Leishmania major, Trypanosoma brucei, Trypanosoma cruzi, Arabidopsis thaliana, Avena sativa, Glycine max, Hordeum vulgare, Oryza sativa, Triticum aestivum, Zea mays, Lycopersicon esculentum, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces bayanus, Saccharomyces castelli, Saccharomyces kluyveri, Saccharomyces kudriavzevii, Neurospora crassa, Magnaporthe grisea, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus, Candida albicans, Coccidioides posadasii, Gibberella zeae, Coprinopsis cinerea, Cryptococcus neoformans, Ustilago maydis and Encephalitozoan cuniculi. In addition, 275 eubacterial and 21 Archaean genomes were searched [see Sinbad-like elements.A panel of fully or partially sequenced entire genomes was searched by BLAST for elements exhibiting sequence similarity to ched see . GenomesSinbad LTR retrotransposon have been assigned accession numbers AY506537, AY506538, AY645721, AAT66412, and AY871176. Other sequences introduced here been assigned GenBank Third Party Annotation accession numbers; BK005570 (Danio rerio), BK005571 (D. rerio), BK005572 (Caenorhabditis briggsae), BK005573 (Anopheles gambiae), BK005574 (D. rerio).Sequences of the MGE, mobile genetic element; ORF, open reading frame; EST, expressed sequence tag; gDNA, genomic DNA; LTR, long terminal repeat; RT, reverse transcriptase; PR, protease; IN, Integrase; CHB, Cys-His box; BAC, bacterial artificial chromosome; MB, megabase pairsCSC carried out the sequence analyses, sequence alignments, phylogenetic studies, other bioinformatics analyses, and Southern hybridizations, participated in cloning, sequencing and design of the study, and, together with PJB, drafted the manuscript. VHM and MEM participated in cloning and sequencing. BHK contributed to the design of experiments and analyses. PJB participated in the design and coordination of the study, and drafting the manuscript. All authors read and approved the final version of the manuscript.Sinbad sequence". Nucleotide and deduced amino acid sequence of the entire Sinbad retrotransposon in BAC clone 33-N-3. Hallmark features of the retrotransposon are identified in colored highlights as described in the key at the bottom of the figure."Annotated Click here for file"RT domain sequences of new and consensus elements used in the phylogenetic analysis". Deduced amino acid sequences of the RT domains used in the phylogenetic analysis from newly characterized elements, uncharacterized elements found within genome survey sequences, and elements for which consensus sequences were used. Accession numbers for the source sequences of each element are listed, as well as references where applicable.Click here for fileSinbad like elements" Table of prokaryotic genomes indicated by whole genome analysis to be devoid of Sinbad like elements."Prokaryotic genomes negative for Click here for file


Family doctors should care for individuals in the context of their family. Family has a powerful influence on health and illness and family interventions have been shown to improve health outcomes for a variety of health problems. The aim of the study was to investigate the Estonian family doctors' (FD) attitudes to the patients' family-related issues in their work: to explore the degree of FDs involvement in family matters, their preparedness for management of family-related issues and their self-assessment of the ability to manage different family-related problems.A random sample (n = 236) of all FDs in Estonia was investigated using a postal questionnaire. Altogether 151 FDs responded to the questionnaire (response rate 64%), while five of them were excluded as they did not actually work as FDs.Of the respondents, 90% thought that in managing the health problems of patients FDs should communicate and cooperate with family members. Although most of the family doctors agreed that modifying of the health damaging risk factors  of their patients and families is their task, one third of them felt that dealing with these problems is ineffective, or perceived themselves as poorly prepared or having too little time for such activities. Of the respondents, 58% (n = 83) were of the opinion that they could modify also relationship problems.Estonian family doctors are favourably disposed to involvement in family-related problems, however, they need some additional training, especially in the field of relationship management. There are significant differences in the way how primary health care is organised in Europe . EstoniaIrrespective of the health system, general practitioners/family doctors should care for individuals in the context of their family . Family In Estonia, a decade has passed after the new speciality, family doctor, was introduced into the health care system. The aim of the present study was to investigate the attitude of FDs in Estonia to family-oriented general practice : FDs' awA 21-item questionnaire was designed for the study. The items were developed by researchers considering the aims of the study.First, the FDs were asked whether their patients have registered on the list by families or not and whether they regard it as appropriate that the family should be cared by one doctor, or whether they think that children should have a separate primary care physician. The FDs rated their opinion on the degree of involvement with various problems in the family: they should deal only with the treatment and counselling of a particular patient, or to cooperate also with family members, in addition to treatment of a particular patient, or they should deal also with emotional and relationship problems of the family members. The questions about FDs' awareness of various issues related to their patients' families such as familial diseases and diseases of family members, financial coping, relationships in the family, living conditions (overcrowding), drug addiction, alcohol abuse, smoking and leisure activities were inquired on a three-step scale : yes, in the case of each patient; yes, in certain cases; no, it is not necessary. FDs' were asked to self-estimate their ability to manage problems in families such as substance abuse and relationship problems. Questions about the FDs' ability to manage problems in families were open-ended, for example: how do you assess your possibilities as a FD to influence relationship problems in the family? If you assess that you cannot influence them, please specify why? If you assess you can influence them, please specify how? Also, the FDs were asked to estimate whether their professional training for dealing with the problems of families is adequate or inadequate. Several questions were related to sociodemographic characteristics (sex, age) and professional history . The questionnaire was piloted for clarity and relevance in a group of five FDs, and minor changes were introduced.rd doctor, in alphabetical order, from the list of doctors who had passed residency or retraining courses in family medicine by that time in Estonia (n = 715). A note of reminder and a new questionnaire was sent to the non-responders 4 weeks after the first mailing. Of the 236 mailed questionnaires, 151 were returned after two mailings (64%), five of them were excluded as the respondents did not actually work as FDs. Thus, altogether 146 questionnaires were included in the study, of these 124 were fully completed, while in 22 cases some of the answers (1 to 3 per questionnaire) were missing.In February 2002, the questionnaire was mailed to 236 FDs. a random sample of family doctors of Estonia. The random sample of FDs was formed by choosing the name of every 3The data were analysed using SPSS for the Windows version 10. The chi-square test was used to test the differences in the proportions, all p-values calculated were two-tailed, the p-values higher than 0.05 were considered non-significant (NS).All open questions were analysed as follows: all statements expressing motivation for or indicating problems with dealing with family issues were marked. Further, all similar expressions were grouped under one category. Proceeding from this, the key problems relevant to the study were identified .The mean age of the respondents was 46 (± 8) years, the mean length of the period during which they had worked in primary health care was 18 (± 9) years and the mean size of the list was 1800 (± 513) patients. Of the respondents 55% worked in urban areas, 40% in rural areas, and 5% worked in both areas. The majority of the doctors (92%) were female. The age and sex distribution of the respondents and non-respondents did not differ significantly.A total of 90 (62%) FDs were of the opinion that it was good to have the same FD for the whole family, 31 (21%) responded that it was preferable that every family member chooses a FD on the basis of personal preference and 25 (17%) thought that children should have a primary health care physician other than adults of the same family. Of the FDs, 119 (82%) responded that most patients were registered in their lists by families.Of the respondents 15 (10%) were of the opinion that FDs should deal only with the health problems of concrete patients without involvement of family members, 94 (65%) responded that, besides managing the health problems of patients, FDs should communicate and cooperate with family members, and 36 (25%) thought that apart from the previously mentioned issues, FDs should deal with the family members' emotional and relationship problems.Over 70% of Estonian FDs agreed that in the case of all patients, it is necessary to be aware of drug addiction in the family, diseases of family members, living conditions and alcohol abuse in the family, while the remainder believed that they should be aware of these issues on certain occasions. Of the respondents, over a third thought that the FD should always be aware of relationships in the family and 12% thought that the FD should always be aware of leisure activities of their patients. However, between 60 to 75% believed that FDs should be aware of these issues in certain occasions  of the respondents felt that their preparedness was adequate. Regarding the other issues, less than half of the respondents considered their training adequate  in families. One hundred (70%) of the respondents reported that this was within the scope of their ability, while the majority (n = 71) stated that the methods used were advice and counselling, but also referral to specialists, use of specific medications and suggestions regarding appropriate reading material. However, 16 FDs admitted that the efficacy of their work in this field was low.Nearly one-third of the respondents (30%) estimated that they were not able to reduce the use of harmful substances in their patients' families. The analysis of the open-ended questions identifed some key problems:• Low motivation of patients.• Socio-economic reasons for substance abuse.• Limited time for consultation.• Inadequate preparedness for management of these issues.One hundred and forty-three FDs responded to the question about their influence on relationship problems in their patients' families, 83 (58%) of them were of the opinion that they were able to modify these problems. In most cases, FDs used advice and counselling (n = 52), but they also cooperated with specialists as the psychotherapist, family therapist, psychiatrist or social worker. Of the family doctors 60 (42%) thought that they were not able to influence the patients' relationship problems. In the analysis of the open questions, the doctors identified several key problems:• Limited time.• Lack of special training.• Patients do not address FDs with their problem.• Patients themselves deny the existence of the problem.• These are the patients' private issues in which physicians could not intervene.versus 57% (n = 24) of those who reported that their preparedness for such issues was not adequate (p < 0.05); in the case of relationship problems, the respective percentages were 41% (n = 34) versus 9% (n = 5) (p < 0.0001). There were found no other significant determinants among the sociodemographic or work related factors.The FDs who were sure that they were able to modify the patients' harmful habits as well as family relationships were more likely to estimate their preparedness for the management of these issues as adequate. Among the doctors who reported that their preparedness for counselling for lifestyle issues was adequate, 76% (n = 76) believed that they were able to treat harmful habits, The present study addressed the family doctors' opinions about their involvement in the patients' family issues. This is the first study of this kind conducted in Estonia, a country where family doctors were introduced into the health care system ten years ago. There is yet no definite agreement as to what are the appropriate, ideal or minimal levels of family orientation that family doctors should have . SeveralOur study revealed that care of patients in the context of the family is an important issue for FDs and that Estonian family doctors have good possibilities to take care of the whole family. Although all patients have the right to choose an individual family doctor, the FDs who responded to the questionnaire were sure that family members mostly have one and the same family doctor. This is concordant with the results of a recent survey among patients according to which 74% of the respondents reported that they had one and the same family physician for the whole family . In EstoIn our study, altogether 90% of the family doctors thought that they should communicate and cooperate with family members in management of the health problems of patients. It is a good result considering that family medicine is a new and developing speciality in Estonia. At the same time, most family doctors are not yet ready to deal with the family members' emotional and relationship problems. Primary care is an important early intervention site of most serious relationship issues, domestic violence, etc . HoweverConcerning the family doctors' attitudes to the importance of awareness of their patients' family-related issues, the results indicated that the awareness of drug addiction, diseases of family members, living conditions of the family and alcohol problems in the family were considered the most essential. The awareness of the patients' relationship problems, economic problems and leisure activities was not so highly valued. A recent survey among patients in Estonia revealed that they were also more disposed to involve the family physician in such problems as harmful habits and diseases in the family, but they were less willing to share the relationship problems . This caThe low willingness to be aware of the patients' relationship problems is partly related to insufficient preparedness in this field, as the doctors who considered themselves to be adequately prepared to tackle relationship issues were also more often willing to do this. From another point of view, family issues, especially relationships and economic situation, are always delicate topics and require consideration of the patient's attitude to corresponding activities. It has been shown that patients vary considerably in their preferences for physician inquiries into such problems as social functioning, psychosocial issues and health risks. Also, it may reflect cultural differences: in the Nordic countries biomedical talk is more common, while in the southern regions psychosocial dialogue is prevalent .Although most of the family doctors agreed that modifying of the health damaging risk factors  of their patients was their task, they also felt that management of these problems was ineffective, or they perceived themselves as poorly prepared, or had lack of time for such activities. This shows that despite the fact that family doctors are becoming increasingly more aware of their role, there exists the actual need to improve their instruments for handling lifestyle related and psychosocial problems. In practice, both individual and family–centered working methods are needed, while the choice depends on the patient's problems and needs . ManagemThe results of the present study allow to conclude that Estonian family practitioners are favourably disposed to involvement in family-related problems, but they need additional training especially in the field of relationship management.The author(s) declare that they have no competing interests.All authors participated in the design of the study. MO and RK carried out the data collection, performed the data analyses and drafted the manuscript. All authors participated in the discussion of the drafts. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Timeliness is a key performance measure of public health surveillance systems. Timeliness can vary by disease, intended use of the data, and public health system level. Studies were reviewed to describe methods used to evaluate timeliness and the reporting timeliness of National Notifiable Diseases Surveillance System (NNDSS) data was evaluated to determine if this system could support timely notification and state response to multistate outbreaks.Published papers that quantitatively measured timeliness of infectious disease surveillance systems operating in the U.S. were reviewed. Median reporting timeliness lags were computed for selected nationally notifiable infectious diseases based on a state-assigned week number and various date types. The percentage of cases reported within the estimated incubation periods for each disease was also computed.Escherichia coli O157:H7 infection, meningococcal disease, salmonellosis, and shigellosis, less than 40% of cases were reported within one incubation period for each reported date type.Few studies have published quantitative measures of reporting timeliness; these studies do not evaluate timeliness in a standard manner. When timeliness of NNDSS data was evaluated, the median national reporting delay, based on date of disease onset, ranged from 12 days for meningococcal disease to 40 days for pertussis. Diseases with the longer incubation periods tended to have a higher percentage of cases reported within its incubation period. For acute hepatitis A virus infection, which had the longest incubation period of the diseases studied, more than 60% of cases were reported within one incubation period for each date type reported. For cryptosporidiosis, Published evaluations of infectious disease surveillance reporting timeliness are few in number and are not comparable. A more standardized approach for evaluating and describing surveillance system timeliness should be considered; a recommended methodology is presented. Our analysis of NNDSS reporting timeliness indicated that among the conditions evaluated (except for acute hepatitis A infection), the long reporting lag and the variability across states limits the usefulness of NNDSS data and aberration detection analysis of those data for identification of and timely response to multistate outbreaks. Further evaluation of the factors that contribute to NNDSS reporting timeliness is warranted. Public health surveillance is defined as the "ongoing systematic collection, analysis, and interpretation of data essential to the planning, implementation, and evaluation of public health practice, closely integrated with the timely dissemination of these data to those who need to know". ReasonsTimeliness is a key surveillance system metric and should be periodically evaluated ,4 becausBefore data can be used for public health action, health-related data must be collected by the public health system, analyzed, and disseminated to those responsible for taking action Figure . Within Each week, states and the U.S. territories report case information on persons suspected of having or diagnosed with a nationally notifiable infectious disease to the Nationally Notifiable Diseases Surveillance System (NNDSS), maintained by the Centers for Disease Control and Prevention (CDC) . A natioInfectious disease surveillance evaluation studies reporting timeliness measures that were published between January 1970 and March 2003 in biomedical and public health literature were reviewed. English-language papers were identified by using the Medline database . The search strategy used various combinations of the following key words "timeliness," "reporting delay," "time delay," "lag time," "disease surveillance," "disease outbreaks," "communicable diseases," and "infectious diseases." Reference lists of the studies identified through the Medline search and studies citing CDC's surveillance evaluation guidelines were also reviewed [Reports were included if they evaluated a public health surveillance system operating in the United States and provided a quantitative estimate of disease-specific timeliness . Studies without quantitative timeliness estimates or that reported a quantitative estimate for a group of infectious diseases (versus a disease-specific estimate) were excluded. In addition, studies describing the timeliness of syndromic surveillance systems were excluded.Information abstracted for the review included the disease(s) under surveillance, the geographic area and time period studied, the purpose of the surveillance evaluation, the surveillance time interval measured, the surveillance processes or actions  from the following list of hierarchical date types: onset date, diagnosis date, date of laboratory result, or date of first report to the community health system. National reporting delay was calculated as the difference in days between the midpoint of the MMWR week and the earliest known date reported in association with the case. This time interval reflects various state-specific surveillance intervals in the surveillance process that occur between the occurrence of a health event and the reporting of that health event to NNDSS, but at a minimum it includes Intervals 1–4 , 30 days for acute hepatitis A virus infection, 4 days for meningococcal disease, 20 days for pertussis, 1.5 days for salmonellosis, and 3 days for shigellosis [To assess whether analysis of NNDSS data could support the timely identification of multistate outbreaks at the national level, the percentage of NNDSS cases reports reported within one to two incubation periods for each of the diseases was determined. Incubation periods were used as a surrogate measure for period of communicability which is critical to consider when implementing effective, disease-specific prevention and control measures. For this analysis, estimated incubation periods were used for the seven nationally notifiable infectious diseases selected for this study: 7 days for cryptosporidiosis, 4 days for gellosis . These dMMWR years 1999, 2000, and 2001.Only finalized case-specific data reported from U.S. states and two autonomous reporting entities (New York City and Washington D.C., referred to as states, hereafter) that designated the reported condition as notifiable (reportable by law or regulation) and that met NNDSS publication criteria  were incNeisseria meningitidis and Haemophilus influenzae infections [Eight papers were identified that met the inclusion criteria for this study  response .Neisseria meningitidis infection surveillance system for 1989–1992 indicated that the lengthy reporting interval limited the usefulness of the system for supporting rapid response for control and prevention [The reporting timeliness of AIDS and bacterial meningitis  surveillance systems were more frequently assessed than those for other infectious diseases. The AIDS reporting timeliness studies indicate that local and national AIDS reporting timeliness meets the goals of the AIDS surveillance systems monitoring trends, targeting prevention programs, estimating needs for medical and social services, and allocating resources ,15,17. Tevention . In contevention . In addievention .The definition of reference dates used in the timeliness evaluations varied. The initial date associated with the case varied among date of disease onset, date of diagnosis, and date of positive culture result. The ending date for the timeliness studies evaluated was the date the case report was received by the public health system, whether at the local, state, or national level. This time period corresponds to the sum of Intervals 1 and 2 or Interval 2 alone for local or state timeliness studies  cryptosporidiosis case reports, 9,674 (78.4%) case reports of E. coli O157:H7 infection, 32,953 (80.3%) case reports of acute hepatitis A virus infection, 5,580 (78.7%) case reports of meningococcal disease, 19,904 (87.5%) case reports of pertussis, 84,746 (70.2%) case reports of salmonellosis, and 41,643 (68.6%) case reports of shigellosis were eligible for analysis. A total of 72,293 (26.4%) case reports were excluded for one or more of the following reasons: reported as a summary or aggregate record in which individual cases may have different event dates (20,194 cases), unknown or missing date types (20,019 cases), date type coded to MMWR report date (11,851 cases), and calculated reporting lag had a value of zero (indicating the event date and midpoint of the MMWR week matched) or had a negative value (indicating the event date was later than the mid-point of the MMWR week [67,557 cases]).For Timeliness of reporting varied by disease and date type Table . For casFor the same date type, NNDSS diseases with longest incubation periods tended to have a higher percentage of cases reported within one or two incubation periods than NNDSS diseases with shorter incubation periods Table . For exaNeisseria meningitidis infection (median 21 days). In addition, the median reporting delay between disease onset and the date of report to CDC was shorter in this study than in a previous study (which used 1987 notifiable disease data) by 10 days for hepatitis A, 5 days for salmonellosis, and 8 days for shigellosis [The 1999–2001 NNDSS meningococcal disease median reporting interval between date of disease onset and date of report to CDC in this study was 8 days shorter than a previous study reported  using 19gellosis .Few published studies evaluating surveillance systems presented timeliness measures. When timeliness was evaluated, standard methods were not used. Information collected by public health surveillance systems should support the quantitative assessment of timeliness by various steps in the pubic health surveillance process. Public health programs should periodically assess timeliness of specific steps in the surveillance system process to ensure that the objectives of the surveillance system are being met. A more structured approach to describing timeliness studies should be considered.Published papers describing local or state surveillance system reporting timeliness generally do not explicitly describe the surveillance system processes contributing to the timeliness measure, such as processing and analyzing the data or implementing a public health action before data are reported from a state to CDC. To facilitate future comparisons of reporting timeliness across jurisdictions, studies should include an explicit description of the public health surveillance reporting process and the surveillance process interval being measured. Additionally, surveillance information systems must support the collection of appropriate reference dates to allow the assessment of the timeliness of specific surveillance processes.A more structured approach to describing timeliness studies could include a description of the following characteristics: 1) the level of the public health system being assessed , 2) the purpose of the surveillance evaluation, 3) goals of the surveillance system, 4) the surveillance interval being measured and a description of the reference dates that define the upper and lower boundaries of the surveillance interval, 5) the surveillance steps (processes or activities) that contribute to the surveillance interval being measured, 6) whether the measured timeliness met the needs of the surveillance step being evaluated, and 7) whether the timeliness met the goals of the surveillance system. No single timeliness measure will achieve the purpose of all evaluations or meet all the goals of the surveillance system. In addition, if the goal of the surveillance evaluation is to identify ways to improve timeliness, the analysis should identify factors associated with delayed reporting, such as the role of specific case ascertainment sources.E. coli O157:H7, meningococcal disease, salmonellosis, and shigellosis). Until reporting timeliness is enhanced, the application of aberration detection analytic methods to NNDSS data to aid in the identification of changes in disease reporting that may indicate a multistate outbreak in time to alert states for the purposes of disease control and prevention may be of limited use. Future work to improve reporting timeliness will need to address the substantial variation across states. As states enhance their reporting mechanisms with the use of automated electronic laboratory reporting systems [The 1999–2001 national notifiable diseases data were timely enough to support the following surveillance objectives: monitoring trends over time, informing allocation of public health resources, monitoring the effectiveness of disease control, identifying high risk populations, and testing hypotheses. If NNDSS data are to be used to support timely identification of and response to multistate outbreaks at the national level, the timeliness of reporting needs to be enhanced for all diseases, but especially for diseases with the shortest incubation periods  cases were excluded from our analysis because the information contained in the database would not permit calculation of timeliness and this exclusion may have resulted in our study results either falsely overestimating or underestimating the magnitude of NNDSS reporting lags.Our study findings are subject to several limitations. The variables available for assessing NNDSS reporting timeliness are based on the MMWR week numbers. Other factors that might have contributed to reporting delay in our study included: the patient's recognition of symptoms; the patient's acquisition of medical care; the use of confirmatory laboratory testing; reporting by the health care provider or the laboratory to the local, county, or state public health authority; the volume of cases identified in the state; case follow-up investigations to verify the case report or to collect additional case information; periods of decreased surveillance system activity due to variable staffing levels; computer system down-time for maintenance, upgrades, or new application development; and data processing routines, such as data validation or error checking. Following a structured approach to evaluation of timeliness by specifying the surveillance objectives and the process(es) being measured may allow better definition of the factors that contribute to reporting delay. It was beyond the scope of this study to assess how these factors contribute to NNDSS reporting timeliness.The reporting timeliness variations across states may result from different reporting protocols in the states  or from variations in how states assign In addition to reporting timeliness, other surveillance system attributes are important to assess (e.g., completeness of reporting). Completeness of notifiable infectious diseases reporting in the United States varies from 9% to 99% . Six of To improve public health surveillance infrastructure and performance in the United States, CDC and local and state health agencies are integrating a number of public health surveillance systems monitoring infectious diseases in the United States, including the NNDSS, into the National Electronic Disease Surveillance System (NEDSS) ,20. NEDSTo facilitate comparisons of surveillance system timeliness studies across jurisdictions or health conditions, a more standardized approach to describing timeliness studies is warranted. Public health surveillance systems should ensure that timeliness can be measured for specific surveillance system processes and in the context of the goals of surveillance. In addition, when timeliness is being measured, it is important to be explicit about how it is being measured. Our analysis of NNDSS reporting timeliness suggests that current acute hepatitis A infection reporting timeliness may be sufficient to support a timely public health response in the event of a multistate outbreak. However, for the other conditions evaluated, the long reporting lag and the variability across states limits the usefulness of NNDSS data and aberration detection analysis of those data for identification of and response to multistate outbreaks. The NNDSS timeliness data presented in this paper represents a baseline against which timeliness can be measured in the future. Further study is needed to identify the major sources of reporting delay and to assess how NNDSS reporting timeliness may be improved for the timely detection of cases and disease clusters.None declared.Both authors contributed equally to project conception and write-up of the manuscript. RAJ was responsible for data analysis. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Published reports quantitatively measuring timeliness of reporting infectious disease surveillance data. Table summarizes the findings of the review of published literature about quantitative measurements of infectious disease surveillance system timelinessClick here for file