People sometimes solve problems with a unique process called insight, accompanied by an “Aha!” experience. It has long been unclear whether different cognitive and neural processes lead to insight versus noninsight solutions, or if solutions differ only in subsequent subjective feeling. Recent behavioral studies indicate distinct patterns of performance and suggest differential hemispheric involvement for insight and noninsight solutions. Subjects solved verbal problems, and after each correct solution indicated whether they solved with or without insight. We observed two objective neural correlates of insight. Functional magnetic resonance imaging  revealed Functional magnetic resonance imaging (fMRI) and electroencephalography (EEG) are used to study neural activity in subjects during a verbal task for which they report solutions achieved by insight Insight is pervasive in human  when he suddenly discovered that water displacement could be used to calculate density. Since then, “Eureka!,” or “Aha!,” has often been used to express the feeling one gets when solving a problem with y animal ) cognitiAlthough many processes are shared by most types of problem solving, insight solutions appear to differ from noninsight solutions in several important ways. The clearest defining characteristic of insight problem solving is the subjective “Aha!” or “Eureka!” experience that follows insight solutions . This suPersistent questions about insight concern whether unconscious processing precedes reinterpretation and solution, whether distinct cognitive and neural mechanisms beyond a common problem-solving network are involved in insight, and whether the apparent suddenness of insight solutions reflects truly sudden changes in cognitive processing and neural activity.Recent work suggests that people are thinking—at an unconscious level—about the solution prior to solving problems with insight. Specifically, while working on a verbal problem they have yet to solve, people presented with a potential solution word read the actual solution word faster than they read an unrelated word . This “sProblem solving is a complex behavior that requires a network of cortical areas for all types of solving strategies and solutions, so solving problems with and without insight likely invokes many shared cognitive processes and neural mechanisms. One critical cognitive process distinguishing insight solutions from noninsight solutions is that solving with insight requires solvers to recognize distant or novel semantic (or associative) relations; hence, insight-specific neural activity should reflect that process. The most likely area to contribute to this component of insight problem solving is the anterior superior temporal gyrus (aSTG) of the RH. Language comprehension studies demonstrate that the RH is particularly important for recognizing distant semantic relations , and bilWe used functional magnetic resonance imaging (FMRI) in (pine, crab, sauce) and attempted to produce a single solution word (apple) that can form a familiar compound word or phrase with each of the three problem words (pineapple, crab apple, applesauce). We relied on solvers' reports to sort solutions into insight solutions and noninsight solutions, avoiding the complication that presumed insight problems can sometimes be solved without insight  of their solutions, “no insight” for 41% (s.d. = 18.9) of their solutions, and “other” for 2% of their solutions. We marked a point about 2 s (rounded to the nearest whole second) prior to each solution button press as the solution event, and examined a time window 4–9 s after this event (i.e., 2–7 s after the button press) to isolate the corresponding hemodynamic response. Solving problems and responding to them required a strict sequence of events , but this sequence was identical whether subjects indicated solving with or without insight, so differences in FMRI signal resulted from the degree to which distinct cognitive processes and neural systems led to insight or noninsight solutions.t < 1.0, p > 0.3). More importantly, the hemodynamic responses to both insight and noninsight solutions in the homologous area of the LH are about equivalent to the response to noninsight solutions in the RH aSTG—it is the strong response to insight solutions in the RH aSTG that stands out. There is no insight effect anywhere within temporal cortex of the LH. At statistical thresholds below significant levels (p < 0.1 uncorrected), there are as many voxels in LH temporal cortex showing a noninsight effect as showing an insight effect.The involvement of the RH rather than the LH for this verbal task is not due to greater difficulty in producing insight solutions: subjects produced insight solutions at least as quickly (mean solution time = 10.25 s, s.d. = 3.58 s) as they produced noninsight solutions (mean = 11.28 s, s.d. = 4.13 s)  as RH aSTG, the event-related signal within it was weak and the insight–noninsight difference (peak difference = 0.15%) was relatively small. (The insight effect may be attributable as much to a negative response for noninsight solutions as to a positive response for insight solutions.)After RH aSTG, the second largest area showing an insight effect in FMRI signal was the medial frontal gyrus in the LH B. AlthouThere also was an insight effect in small clusters in or near bilateral amygdala or parahippocampal gyrus. Again, regional signal was low (83% of the brainwide average), and the signal difference was small (peak = 0.16%). However, an amygdalar response may be expected, given the emotional sensation of the insight experience (Parsons and Osherson, 2001). Hippocampal or parahippocampal involvement is also plausible, if memory interacts with insight solutions differently from how it interacts with noninsight solutions. For instance, insight problems may encourage distinct memory encoding  or may rSeveral cortical areas showed strong solution-related FMRI signal, but approximately equally for insight and noninsight solutions. Some of these areas (e.g., motor cortex) relate to the response sequence rather than solution processes; other areas probably reflect component processes of a problem-solving network common to both insight and noninsight solving, such as retrieving potential solutions. Two areas that may be of interest for future studies are AC and posterior middle/superior temporal gyrus. Both these areas, in the RH only, showed strong, negative solution-related signal, approximately equal in the two solution types. AC is an area that might be predicted to be involved in reorienting attention as solvers overcome impasses, given its role in performance monitoring and cognitive control . RH postA separate group of subjects participated in fundamentally the same paradigm while we continuously recorded EEGs from the scalp. We then compared time-frequency analyses of the EEGs associated with insight solutions versus noninsight solutions. EEG provides temporal resolution greatly superior to that of FMRI and thus can better elucidate the time course and suddenness of the insight effect. Furthermore, complex EEG oscillations can be parsed into constituent frequency components, some of which have been linked to particular types of neural and cognitive processes .The high temporal resolution of EEG allows us to address one of the fundamental questions raised earlier: does insight really occur suddenly, as subjective experience suggests? For problems typically solved without insight, solvers report gradually increasing closeness to solution. In contrast, for problems typically solved with insight, solvers report little or no progress until shortly before they actually solve the problem . SimilarWe predicted that a sudden change in neural activity associated with insight solutions would produce an EEG correlate. Specifically, we predicted that high-frequency EEG oscillations in the gamma band (i.e., greater than 30 Hz) would reflect this sudden activity, because prior research has associated gamma-band activity with the activation of perceptual, lexical, and semantic representations . Gamma-bt[18] = 3.47, p=0.003); there was no difference in mean response times .Participants solved 46% (s.d. = 8.2) of the problems correctly within the time limit. Of correctly solved problems, subjects reported more insight solutions (56%, s.d. = 8.4) than noninsight solutions (42%, s.d. = 9.0), (F[1,18] = 6.68, p = 0.019) and insight × time window × Hemisphere (F[1,18] = 8.11, p = 0.011) interactions. The overall interaction occurred because there was an insight × hemisphere interaction from −0.30 to −0.02 s (F[1,18] = 4.61, p = 0.046) but no effect in the −1.52 to −0.36 s time window. Within the −0.30 to −0.02 s interval for these two electrodes, there was a significant insight effect at the right temporal (T8) site (t[18] = 3.48, p = 0.003), but not at the homologous left temporal (T7) site or any other LH temporal electrode. Laplacian mapping of this effect (There was a burst of gamma-band activity associated with correct insight solutions (but not noninsight solutions) beginning approximately 0.3 s before the button-press solution response at anterior right temporal electrodes , with nos effect B is remaThe gamma burst in the right temporal area cannot be attributed to motor processes involved in making the response because (A) motor activity associated with the bimanual button press would have caused a bilateral gamma burst, not a unilateral one; (B) the location of the gamma burst as determined by Laplacian mapping B is not Other planned statistical tests (ANOVAs) examined possible insight-related frontal theta (5–8 Hz), posterior alpha (8–13 Hz), and fronto-central beta (13–20 Hz) activity. There were no statistically significant theta or beta effects.  There was a significant posterior alpha effect, which is discussed below.Complex problem solving requires a complex cortical network to encode the problem information, search memory for relevant information, evaluate this information, apply operators, and so forth. The FMRI and EEG results reported here conclusively demonstrate that solving verbal problems with insight requires at least one additional component to this cortical network, involving RH aSTG, that is less important to solving without insight. The insight effect in RH aSTG accords with the literature on integrating distant or novel semantic relations during language comprehension. When people comprehend (read or listen to) sentences or stories, neural activity increases in aSTG or temporal pole bilaterally more than when comprehending single words . Neural Like the results in language processing, the current results are predicted by the theory that the RH performs relatively coarse semantic coding . This thWe suggest that semantic integration, generally, is important for connecting various problem elements together and connecting the problem to the solution, and that coarsely coded semantic integration, computed in RH aSTG, is especially critical to insight solutions, at least for verbal problems . People come to an impasse on insight problems because their retrieval efforts are misdirected by ambiguous information in the problem or by their usual method for solving similar problems. Large semantic fields allowing for more overlap among distantly related concepts  may help overcome this impasse. Because this semantic processing is weak, it may remain unconscious, perhaps overshadowed by stronger processing of the misdirected information , and solA persistent question has been whether the cognitive and neural events that lead to insight are as sudden as the subjective experience. The timing and frequency characteristics of the EEG results shed light on this question. We propose that the gamma-band insight effect in Suddenly recognizing new connections between problem elements is a hallmark of insight, but it is only one component of a large cortical network necessary for solving problems with insight, and recognizing new connections likely contributes to other tasks, such as understanding metaphors  and deriF[2,36] = 4.13, p = 0.027, with the Huynh-Feldt correction). Follow-up t-tests in each time window yielded significant effects of insight in the first time window at both electrode sites  and in the second time window only at the RH site (PO8: t[18] = 2.17, p = 0.043), with a reversal of the direction of the effect. The third time window yielded no significant effects.We turn now to another result from the EEG time-frequency analysis, which was not predicted but nevertheless suggests a provocative interpretation. The gamma burst thought to reflect the transition of the insight solution from an unconscious to a conscious state was preceded by insight-specific activity in the alpha band (8–13 Hz). Specifically, there was a burst of alpha power (estimated at 9.8 Hz) associated with insight solutions detected over right posterior parietal cortex from approximately 1.4 s until approximately 0.4 s before the solution response, at which point insight alpha power decreased to the level of noninsight alpha power, or below . An ANOVAlpha rhythms are understood to reflect idling or inhibition of cortical areas . IncreasThis interpretation of the early insight-specific alpha effect is consistent with previous behavioral research suggesting that, prior to an insight, the solution to a verbal problem can be weakly activated , especiaIn sum, when people solve problems with insight, leading to an “Aha!” experience, their solutions are accompanied by a striking increase in neural activity in RH aSTG. Thus, within the network of cortical areas required for problem solving, different components are engaged or emphasized when solving with versus without insight. We propose that the RH aSTG facilitates integration of information across distant lexical or semantic relations, allowing solvers to see connections that had previously eluded them. In the two millennia since Archimedes shouted “Eureka!,” it has seemed common knowledge that people sometimes solve problems—whether great scientific questions or trivial puzzles—by a seemingly distinct mechanism called insight. This mechanism involves suddenly seeing a problem in a new light, often without awareness of how that new light was switched on. We have demonstrated that insight solutions are indeed associated with a discrete, distinct pattern of neural activity, supporting unique cognitive processes.Ten men and eight women were paid to participate in Following practice, subjects attempted 124 compound remote associate problems during FMRI scanning. These problems b can be Prior to the experiment subjects were told the following: “A feeling of insight is a kind of ‘Aha!' characterized by suddenness and obviousness. You may not be sure how you came up with the answer, but are relatively confident that it is correct without having to mentally check it. It is as though the answer came into mind all at once—when you first thought of the word, you simply knew it was the answer. This feeling does not have to be overwhelming, but should resemble what was just described.” The experimenter interacted with subjects until this description was clear. This subjective rating could be used differently across subjects , blurring condition boundaries; yet the distinct neural correlates of insight observed across the group demonstrate that there was some consistency.If subjects failed to solve problems within 30 s, the “Solution?” prompt appeared, and subjects pressed the “no” buttons and verbalized “Don't Know.” Then the “Insight?” prompt appeared, and subjects pressed the “no” buttons again. After the insight rating, subjects performed three line-matching trials (3 s each) to distract them from thinking about the problems, allowing the critical BOLD signal to return to baseline . The totImaging was performed at the Hospital of the University of Pennsylvania, on a 1.5 Tesla GE SIGNA scanner with a fast gradient system for echo-planar imaging and a standard head coil. Head motion was restricted with plastic braces and foam padding. Anatomical high-resolution T1-weighted axial and sagittal images were acquired while subjects performed practice trials. Functional images  were acquired in the same axial plane as the anatomical images using gradient-echo echo-planar sequences sensitive to BOLD signal . Each fu3.Images were coregistered through time with a three-dimensional registration algorithm . Echo pl3 in volume  in which each voxel was reliably different across subjects, (t[12] > 3.43, p < 0.005 uncorrected). Monte Carlo simulations with similar datasets reveal low false positive rates with these criteria. RH aSTG was the only cluster to exceed these criteria, and converging evidence and the a priori prediction about RH aSTG strengthen confidence in this result.Data were analyzed using general linear model analysis that extracted average responses to each trial type, correcting for linear drift and removing signal changes correlated with head motion. Each TR was divided into two 1-s images to improve time locking of the solving event and the functional image data  from 128 tin electrodes embedded in an elastic cap (linked mastoid reference with forehead ground) placed according to the extended International 10–20 System. Prior to data analysis, EEG channels with excessive noise were replaced with interpolated data from neighboring channels. Eyeblink artifacts were removed from the EEG with an adaptive filter separately constructed for each subject using EMSE 5.0 . Induced oscillations were analyzed by segmenting each subject's continuous EEG into 4-s segments beginning 3 s before each solution response. 0, in the time domain has the formTime-frequency transforms (performed with EMSE 5.0) were obtained by the application of complex-valued Grossmann-Morlet wavelets, which are Gaussian in both time and frequency. Following 0 is a nondimensional frequency. In this case, ω0 is chosen to be 5.336, so that ∫ϕ0(t) ≅ 0. The constant π−¼ is a normalization factor such that ∫(ϕ0(t))2 = 1. For the discrete time case, a family of wavelets may be obtained aswhere ωt is the sample period (in seconds), s is the scale (in seconds), and n is an integer that counts the number of samples from the starting time. The Fourier wavelength λ is given bywhere δIn the frequency domain, the (continuous) Fourier transform of wheree-folding time and frequency may serve as quantitative measures of dispersion. Note that these dispersions are a function of the scale, s. For a selected frequency, 𝒻c = 1/λ, or from One reasonable way to measure the “resolution” of the wavelet transform is to consider the dispersion of the wavelets in both time and frequency. Since the wavelets are Gaussian in both domains, the e-folding time is c. From e-folding frequency is e-folding time is 0.12 s and the e-folding frequency is 2.6 Hz. For a 40-Hz ( gamma-band) center frequency, the e-folding time is 0.03 s and the e-folding frequency is 10.5 Hz. Note that these e-folding parameters imply that wavelet scaling preserves the joint time-frequency resolution , with higher temporal resolution but broader frequency resolution as the wavelet scale decreases.Then substituting into Segments corresponding to trials for which individual subjects produced the correct response were isolated and averaged separately according to whether or not the subject reported the experience of insight. Planned statistical tests (repeated-measure ANOVAs) were performed in order to detect insight-related effects on frontal midline theta (5–8 Hz), posterior alpha (8–13 Hz), fronto-central beta (13–20 Hz), and left and right temporal gamma (20–50 Hz). Response-locked event-related potentials (ERPs) were also computed using the same analysis epoch. Standard ERP analyses yielded no evidence of statistically significant effects, likely because ERPs reflect phase-locked activity rather than the induced (i.e., nonphase-locked) activity examined in the wavelet analyses; due to the long response times evident in this experiment, phase locking resulting from problem presentation would not be expected.EEG effects were topographically mapped by employing spline-based Laplacian mapping with an FMRI-derived realistic head model and digitized electrode positions. Localization of EEG/ERP signals is a form of probabilistic modelling rather than direct neuroimaging. In contrast to other techniques, source estimation by Laplacian mapping indicates the presence of superficial foci of neuroelectric activity with minimal assumptions.Figure S1The far left lane shows for each region a single slice best depicting the cluster activated above threshold; middle lane shows time course of signal following insight (red line) and noninsight (blue line) solutions, across the entire active cluster; right panel shows the “insight effect” .t[12] = 2.83, p < 0.015); (B–D) depict clusters of FMRI signal at the same t-threshold used in the main paper (t[12] = 3.43, p < 0.005), but the clusters are too small to surpass cluster criterion.(A) depicts bilateral IFG with lowered threshold ((B) LH medial frontal gyrus;(C) LH PC gyrus;(D) LH amygdala . Spatial coordinates and other are details listed in (914 KB PDF).Click here for additional data file.


PLoS Medicine editors very useful [I find the arguments raised by the y useful  as I hadIt is not only colleagues in research and allied professions who need access but the global community, including members of the public wherever they live, those who participate in trials and those who will be on the receiving end of their outcomes.The annual reports of research ethics committees (RECs) are supposedly in the public domain after approval by Strategic Health Authorities in the UK. But very few members of the public know of their existence or how to access them. Approaches to individual committees even now can meet with varied reactions, from suspicious, defensive, or hostile—reluctantly sending one report, quizzing as to which organisation the enquirer belongs to and why they should want one—to extremely welcoming of interest and discussion.The annual reports should be easily accessible online by now, surely, but they are not. The activities of RECs and information on what research is being carried out in the name of society as a whole largely remain hidden from public view.www.corec.org.uk) or OREC . COREC has not been open about dealing with issues of concern raised with them in the past. They do state that public interest is welcome now, so it would show a real commitment to making research activity more open if they would show support for totally open access to a register and to promote that through their Web site.There is no information about public access on COREC (Central Office for Research Ethics Committees;


In its extreme form, methylation is involved in silencing one of the two X chromosomes in female mammals. Aberrant methylation underlies susceptibilities to several forms of cancer, and is likely to be involved in numerous other human diseases.For a recipe to become a meal, it's often necessary to embellish or modify the basic instructions—and to keep a note of the changes that work, so that it can be just as delicious next time around. The same is true for a gene, whose basic recipe—its nucleotide sequence—can be heritably annotated to “epigenetically” influence its level of expression without altering its sequence. Among the many epigenetic influences at work in the genome, methylation of cytosine is one of the most versatile and powerful. Addition of a methyl  is to map the methylation patterns of human genes, and to determine how they vary: among individuals, among tissues within an individual, and even over time within a single tissue. In this issue, Stephan Beck and colleagues describe the execution and results of a HEP pilot project, in which they analyzed methylation within the major histocompatibility complex (MHC), the set of genes that establish an individual's self-identity within the context of immune surveillance.The key to any such large-scale project is high throughput—a rapid, efficient set of technologies that produce the needed data with minimal human intervention. The strategy used by Rakyan et al. included bisulfite sequencing of DNA, in which unmethylated cytosines are chemically converted to uracils, while methylated cytosines are not. Software they developed detects the methylated sites and provides an overall measure of the methylation level within any given sequence. They confirmed the accuracy of their method with mass spectrometry, an alternative method also suitable for high-throughput screening.They initially analyzed 253 sequences within 90 genes in the MHC, about two-thirds of the total, from multiple tissues in multiple individuals. They found that most genes were either completely methylated or completely unmethylated, while relatively few had an intermediate value. The significance of this distribution pattern is not yet clear, although it does confirm similar results in smaller samples from other research groups. The researchers also confirmed that so-called CpG islands, regions rich in CG dinucleotides, are relatively hypomethylated, especially when they occur at the upstream end of a gene.Rakyan et al. also found differences in methylation levels among tissues, with some suggestion that the variations influence tissue-specific alternative splicing, at least in some genes. Intriguing inter-individual differences were also found, with median methylation levels differing significantly between individuals for at least one tissue at almost half the sites analyzed. For instance, such differences were found in liver for the regulatory region for the tumor necrosis factor gene.A major goal of the HEP is to identify methylation variable positions, sites whose methylation state is linked with some important biological state, be it tissue type, developmental stage, or disease state. The pilot project described here begins this undertaking, which will be greatly expanded as the HEP progresses. The first phase of the full-scale HEP, an analysis of 5,000 DNA sequences, is currently underway.


We compared two methods of rooting a phylogenetic tree: the stationary and the nonstationary substitution processes. These methods do not require an outgroup.Given a multiple alignment and an unrooted tree, the maximum likelihood estimates of branch lengths and substitution parameters for each associated rooted tree are found; rooted trees are compared using their likelihood values. Site variation in substitution rates is handled by assigning sites into several classes before the analysis.In three test datasets where the trees are small and the roots are assumed known, the nonstationary process gets the correct estimate significantly more often, and fits data much better, than the stationary process. Both processes give biologically plausible root placements in a set of nine primate mitochondrial DNA sequences.The nonstationary process is simple to use and is much better than the stationary process at inferring the root. It could be useful for situations where an outgroup is unavailable. Typicalet al. , a nonrestationary, i.e., the sequence composition is unchanged in time, and is equal to the equilibrium distribution of the rate matrix Q. The consensus is that it does not have enough power to discriminate among the candidate rooted trees. In this paper, we investigate a slightly more general, nonstationary process: in which the initial distribution π may not be the equilibrium distribution of the rate matrix Q. A priori, giving up stationarity is expected to produce a much better fit to data, since sequence composition is known to evolve, and should be accounted for. Indeed, substitution models where each branch has its own rate matrices had been used to resolve deep splittings in certain phylogenetic trees; see Yang and Roberts, and Galtier and Gouy [π and Q. The rooted trees are then ranked in descending order of likelihoods. We model systematic variation in substitution rates among sites by assigning sites into several classes, and the relative rate for each class is estimated by ML; this is equivalent to the combined analysis framework of Yang [The nonreversible substitution process, introduced by Yang , is statand Gouy ,7. Our p of Yang .xenopus laevis). The analyses were based on all available mitochondrial protein-coding genes, as well as two nuclear protein-coding genes. Next, we applied the methods to a set of primate mitochondrial DNA sequences.We compared the ability of the stationary and nonstationary processes to place the root in three groups of species where the answer is considered well-known: (1) human, chimpanzee and gorilla, (2) human, chimpanzee, gorilla and orangutan, (3) human, mouse, chicken and frog  human, chimpanzee and gorilla, (2) human, chimpanzee, gorilla and orangutan, and (3) human, mouse, chicken and frog (xenopus laevis). The sequences were downloaded from Genbank and aligned using the CLUSTALW alignment of the amino acid sequences. Most alignments looked quite solid [see Additional files]. The beginning of the alignments for the genes COX1, CYTB, ND1 and ND6 were slightly adjusted. The root positions are assumed to be on the (1) gorilla, (2) orangutan, and (3) frog branch, respectively. The branches on a tree are referred to by the organism names, except for the case of four taxa, where there is an internal branch , stationary (STA) and reversible (REV) substitution models to all available mitochondrial protein-coding genes, as well as the nuclear genes ganisms:  human, cIn group 1, the NONSTA and STA processes correctly placed the root in 8 and 6 genes respectively, out of 13 genes Table . In groualbumin and c-myc and three mitochondrial genes, COX1, COX2 and ATP6 from group 3 (with some mouse genes replaced with rat genes) were studied by Huelsenbeck et al. [ATP6, with NONSTA again noticeably more discriminative.The nuclear genes k et al. . For theet al. and Yang [Brown and Yang ,9 studieOur results confirmed earlier findings that the stationary process (STA) is not very good at discriminating among rooted trees corresponding to the same unrooted tree. In contrast, the nonstationary (NONSTA) process seems much more effective, with individual genes, and with combined genes. It is quite clear that the difference in log likelihoods between fitting STA and the reversible process (REV) is often small, and statistically insignificant, based on the likelihood ratio test, while those between NONSTA and STA, and between NONSTA and REV, are often large, and statistically very significant. Though the chi-square distribution may be inappropriate , it seemet al.'s analysis using STA failed to place the root correctly in any of the genes albumin, c-myc, COX1, COX2 and ATP6, there are some differences between the analyses. The raw data were different: the rat albumin and c-myc genes were used by Huelsenbeck et al.; since mouse and rat are very similar, this is not likely to matter much. Secondly, the alignments were probably different, though since the sequences are quite similar, this should not be too important. It is plausible that most of the discrepancies between the results is due to the difference in the estimation procedure (maximum likelihood vs. Bayesian) and to the fact that in Huelsenbeck et al., site variation was modeled by the gamma distribution [Although Huelsenbeck ribution , whereasalbumin, c-myc and ND2, showing the usefulness of the third codon position in this dataset, despite its markedly higher substitution rates. We also found that the pairwise identity at the third codon positions for all genes in groups 3 ranges from 34% to 61%. Base composition being generally nonuniform, the expected pairwise identity at saturation (i.e., infinite evolutionary distance) is lower than 25%. This seems to indicate that the third codon position is not saturated, and hence the phylogenetic information from this position is not just the base composition at each taxon. In addition, the base composition at the third codon position for some genes is quite different from the other positions. Our model does not fit these genes as well as a model where separate processes are associated with the codon positions. Such a model will be investigated in future.Estimates of the relative rates are quite independent of the model used, and their relative magnitudes are largely within expectations. In particular, for group 3, the relative rates for codon positions 1, 2, and 3 fall between .2 and 1.1, .1 and .6, and 1.5 and 2.7 respectively. For all genes, the third codon position evolved the fastest, followed by the first and second positions. To gauge the contribution from the third codon position, we left out the corresponding bases in group 3 and reran the analysis with NONSTA. This gave the correct root placement in only three genes: The NONSTA process is only slightly more complicated to apply, compared to the STA and REV processes. The fact that it works quite well in the verification studies and predicts biologically plausible roots for the nine-primate data demonstrates its utility and perhaps argues for its use in routine phylogenetic analysis. In any case, if no suitable outgroup is available, it could be worthwhile to try it. Though the NONSTA process is the most general time-homogeneous Markov process, it is still simplistic and imposes a severe constraint on the evolution of base composition: if two leaf nodes are at the same distance from the root, then the process stipulates that the corresponding sequences must have the same composition. This is patently unrealistic: once lineages split, they should evolve quite independently, and may explain the failure of the process at estimating the root placement for some genes. However, it is still valuable even if it does not always work, in that it can serve as a base from which exploration of richer models can be launched. For instance, one could identify lineages where the evolution significantly deviates from expectations, and then allow these lineages to have different rate matrices, which brings us closer to the very rich models of ,7,13,14.The nonstationary substitution process is simple to use, has much greater power at estimating the root compared to the stationary process, and also fits data much better than the stationary and reversible processes. It seems feasible to use this process in analyses where a suitable outgroup is not easily available. It is also a good starting point for conducting more sophisticated phylogenetic analysis with richer models.Substitutions in DNA sequences are assumed to occur independently at each site according to a Markov process, i.e., given the present base, future substitutions are independent of past substitutions.a to b is the (a, b)-entry in a 4 × 4 rate matrix Q; the diagonal entries are such that each row sums to 0. For any t > 0, the transition probability P(t) is given by P(t) = exp(Qt). Let π be a probability distribution on the DNA bases. The pair (π, Q) defines a substitution process on a rooted tree, as follows: pick a base at the root according to π, then run the substitution process according to Q down the tree, splitting into independent copies whenever a branching is encountered. The joint probability of the observed bases at the leaf nodes can be computed using almost exactly the same algorithm by [Furthermore, it is assumed that the process is time-homogeneous, i.e., substitution rates stay constant in time. As usual, the substitution rate from base rithm by .π, Q). Associated with the rate matrix Q is a unique distribution πQ, called the equilibrium distribution of Q, such that the matrix product πQ × Q is the zero vector. The process (πQ, Q) is stationary, i.e., the sequence composition remains unchanged through time, and is described by πQ. Q is said to be reversible if it satisfies the detailed balance condition:There are two important special cases of the time-homogeneous process  is then reversible, i.e., statistically the process looks the same in forward and backward time. In particular, as shown in [et al. [π and 12 off-diagonal entries in Q), 12  and 9 . Since the models are nested, the likelihood ratio test can be used to assess the relative goodness-of-fit of the MLEs. It is standard practice to allow only calibrated rate matrices, i.e., Q satisfieswhere Πshown in , the joishown in ,16,17. Sshown in ,18-22. Tshown in , and sub [et al. . Yang reπ, Q), with calibrated Q, since in general π ≠ πQ, it is not true that the expected number of substitutions in 1 time unit is 1, but the difference gets arbitrarily small as time goes to infinity.so that a branch length is the average number of substitution events per site. We adopt this practice, and remark that for the nonstationary process (i an unknown positive number ri, with the constraint thatThe sites in a DNA sequences can have very different substitution rates, the most well-known example being coding sequences, where the third codon positions evolved much faster than the others because of the degeneracy of the genetic code. In cases where the assignment of sites into several classes is known in advance, such as a coding sequence, the easiest way to deal with it is to associate to class ni is the number of sites in class i. The relative rate ri either expands or shrinks the tree depending on whether it is more or less than 1. The constraint gives a new interpretation of a branch length: it is now the average over all sites of their expected number of substitutions. Thus, this approach is similar to [where milar to : effectimilar to  or the hmilar to .Given a rooted tree relating aligned coding sequences, we seek the ML estimates of the branch lengths, the substitution parameters, and the relative rates. For other sequences, the relative rates are not estimated. Gradient-based methods are perhaps the most efficient at finding the maximum. The EM algorithm  is anothThe estimation procedure was implemented in C, and the source code can be requested from the first author.The idea was conceived by the first author and was inspired and refined by the second author. The first author composed the code and performed the data analysis.A text file containing the amino acid sequence alignments for group 1.Click here for fileA text file containing the amino acid sequence alignments for group 2.Click here for fileA text file containing the amino acid sequence alignments for group 3.Click here for file


Malaria remains one of the greatest threats to global health, infecting more people than ever before. Confined mainly to the tropical areas of Africa, Asia, and Central America, malaria hits Africa the hardest; the poverty-stricken lands of sub-Saharan Africa account for 90% of malaria infections worldwide. Despite ongoing efforts to battle the disease—by controlling mosquito populations, reducing human contact, and developing drug prevention and treatment—the crisis continues to worsen.The primary variables affecting risk of infection are the rate at which humans are bitten and the proportion of mosquitoes that are infectious. These two factors are often regarded as positively correlated, meaning that if the percentage of infectious mosquitoes increases, so will the human biting rate. But in a new study, David Smith, Jonathan Dushoff, and F. Ellis McKenzie challenge this assumption. Using a mathematical modeling approach to examine the relative contributions of the two factors across different landscapes and seasons, the authors show that the factors are not positively correlated. In fact, their calculations show that the rate humans are bitten and the proportion of infectious mosquitoes peak at different times and places.Their modeling results suggest that the standard metric to estimate risk of infection—the number of times an infectious mosquito bites a person per day, called the entomological inoculation rate (EIR)—is flawed when variable conditions are taken into account. Using the average EIR to estimate average risk of infection in variable environments generates biased estimates because there is not a direct correlation between EIR and the proportion of humans who are infected.The distribution of humans and suitable habitat for mosquito larvae varies across the landscape. And the density of mosquito populations varies seasonally, rising and falling with changes in rainfall, temperature, and humidity. Temporal and spatial variations in mosquito populations affect the rate humans get bitten, the number of infectious mosquitoes, and the risk of infection. To understand how these space- and time-induced variations in mosquito populations shape the epidemiology of human infection, Smith and colleagues developed a set of mathematical models that calculate the relative impact of different parameters, in order to determine which factors most influence where and when risk of infection is highest.First, they evaluated what factors affect the primary components of the EIR: the human biting rate and the proportion of infectious mosquitoes. As expected, the model predicts that fluctuations in mosquito density influence the EIR by changing the human biting rate. As more people are bitten, more people become infected; consequently, more mosquitoes feed on infected humans and so become infectious. Only adult mosquitoes transmit infection, so as mosquito populations age, the proportion of infectious mosquitoes increases. During the dry season, few mosquitoes are born, and so while the human biting rate and EIR decline, the proportion of infectious mosquitoes increases.Because mosquito populations are densest near breeding sites—where younger mosquitoes outnumber adults—the human biting rate and the number of bites by infectious mosquitoes per person per day reflect shifts in mosquito density, not in the proportion of infectious mosquitoes. The model predicts that human biting rate is highest shortly after mosquito population density peaks, typically either near breeding sites or where human density is highest. The proportion of infectious mosquitoes, on the other hand, reflect the age of the mosquito population: it peaks where older mosquitoes are found—farther from breeding sites—and when populations are declining.By mapping larval habitats against the local risk of mosquito-borne infections, Smith and colleagues conclude, epidemiological models can be developed to predict risk for local populations. Their results make the case that mathematical models can help public health officials calculate risk of infectious diseases in heterogeneous environments—that is, real world conditions—when vector ecology and the parameters of transmission are well characterized. Any plan to prevent and control the spread of mosquito-born infections would clearly benefit from paying attention to mosquito demography and behavior.


Modeling latent variables such as physical disability is challenging since its measurement is performed through proxies. This poses significant methodological challenges. The objective of this article is to present three different methods to predict latent variables based on classical summed scores, individual item responses, and latent variable models.This is a review of the literature and data analysis using "layers of information". Data was collected from the North Carolina Back Pain Project, using a modified version of the Roland Questionnaire.The three models are compared in relation to their goals and underlying concepts, previous clinical applications, data requirements, statistical theory, and practical applications. Initial linear regression models demonstrated a difference in disability between genders of 1.32 points (95% CI 0.65, 2.00) on a scale from 0–23. Subsequent item analysis found contradictory results across items, with no clear pattern. Finally, IRT models demonstrated three items were demonstrated to present differential item functioning. After these items were removed, the difference between genders was reduced to 0.78 points (95% CI, -0.99, 1.23). These results were shown to be robust with re-sampling methods.Purported differences in the levels of a latent variable should be tested using different models to verify whether these differences are real or simply distorted by model assumptions. Clinical researchers frequently use statistical models in an attempt to model outcomes that are not directly measured, also known as latent variables. Examples of such latent variables include mental health, quality of life, and physical disability. Although groups of items (questions) known as outcome scales can be assumed to measure latent variables, it is methodologically challenging to aggregate item responses into scores that accurately and reliably represent the latent variable.The aim of this study is to point that the choice of models with biased assumptions can lead to different conclusions regarding the associations between latent variables and predictors. Three alternative methods are presented: Prediction of latent variables measured as summed scores using linear regression models, prediction of individual item responses using logistic regression models and propensity scores to control for differences in item responses, and prediction of latent variables using Item Response Theory models with covariates. Since all three methods are statistically sophisticated, they will be described using the technique of "layers of information", and used to evaluate the purported association between gender and disability. Specifically, we will test whether this association can be explained by different reporting patterns.The method of "layers of information" was designed to explain complex statistical methods to audiences with a variety of previous quantitative backgrounds. Each layer is associated with a progressive level of complexity; thus, ensuring that readers with different needs can understand the technique to a level that will enable them to at least understand the statistical method of a clinical study (first layer) and ultimately to apply the statistical method to a new research study (last layer). In the current study, we have used five layers of information: (1) General description, (2) Examples of previous clinical applications, (3) Data requirements, (4) Statistical Theory, and (5) Analysis and Reporting.A latent construct is a concept not directly measured, but that can be estimated through proxy measures. Physical disability is an example since its level is frequently inferred from responses given to a series of items in an outcomes scale measuring patients' ability to perform activities of daily living. Because latent variables cannot be directly measured and predicted, several statistical techniques were devised to approach this problem Figure .The most common approach is to simply add patients' responses to each item; thus, creating a summed score. Summed scores are then used to determine significant predictors in a regression model Figure .Two assumptions underlie this strategy. First, we assume that the contribution of each item to the latent variable is known. For example, in a disability scale where patients are questioned about their ability to "raise a glass of water" and to "raise a 40-pound bag", researchers assume that they know the exact amount of disability associated with each of the activities stated by these items. In a scale that does not discriminate between the level of disability associated with each item, the assumption would be that answers to each of these items would represent the same amount of disability, when, in fact, they may not.The second assumption when using summed scores is that each item measures the latent construct without any interference from extraneous factors. For example, it is assumed that two individuals with the same neck disability level, but different educational levels would have similar answer patterns for an item such as "I feel pain in my neck after reading for more than two hours". In this example this assumption might not be true since individuals with different educational levels may have different levels of exposure to a two-hour reading session and consequently have a different perception of the disability caused by such activity. Therefore, in spite of having the same disability level, they would probably provide different answers to the same item. This phenomenon is known as Differential Item Functioning, previously known as item bias.A second approach is to use answers from each item and then determine how each predictor is associated with individual item responses Figure . AlthougThe last and most recent approach is to use statistical models that will concomitantly determine the latent construct level and its association with the predictor of interest Figure . The maiIn a study designed to predict factors associated with post-treatment disability after lower-extremity soft tissue sarcoma, Davis  calculatIn a study evaluating the prediction of visual disability based on individual objective measures of visual impairment, Bandeen-Roche  regresseAlthough these models bring new insights into the association between individual physical activities and their respective predictors, they cannot clarify whether these were true predictors or whether they simply presented different reporting patterns.To our knowledge, although multiple previous clinical research projects have used IRT for the determination of scale scores , no prevFirst, a latent construct has to be measured through a set of proxy variables. These indicators may have responses in various formats, including dichotomous (yes/no), ordinal (e.g., a little, moderate, a lot), or nominal . IRT models assume that the latent construct is continuous and, in most cases, unidimensional, meaning that one single latent construct is assumed.Predictors can be continuous or categorical variables.Previous studies have estimated that, for logistic regression models, one should have at least 10 events per predicting variable , while fy using a linear combination of predictor variables xj, where j represents several predicting variables 1, 2, ..., p. It is assumed that no missing values are present for every observation. The fitted values, or predicted summed scores, are then the sum of coefficients βj multiplying each of the xj plus an intercept β0, although the later may be absent in some models. This model can be represented by:Differences in summed scores according to a set of predictor or covariates can be described using linear regression. In these models, the summed score is represented by y - β0 + β1 x1 +...+ βp xpith of n observations are yi, xi1 ,..., xip, then the fitting criterion chooses the βj to minimize:Ordinary least-squares models estimate the coefficients to minimize the squared sum of residuals. If the response and predictors corresponding to the ith observation as:The standard statistical theory of linear models makes the first formula more explicit by writing the model for the ci are independently and identically distributed; the ci have mean zero and finite variance σ2; the ci have a normal distribution.This model makes the following assumptions: The Individual responses to dichotomous items can be predicted by generalized linear models using a binomial distribution and, most commonly, a logit link function that will bound the probability of an answer to be between 0 (answer = no) and 1 (answer = yes). The logit link can be expressed by:π is the probability of a positive answer andx is a vector with item responses . To linearize the function, the dichotomous response for each item can be algebraically transformed to:where p(0|x), where 0 is the latent construct.Notice that, in contrast to linear models, the logistic model does not have an error term since it models the probability of an event directly that will determine the variability of the binary outcome. Logistic models are estimated by maximum likelihood, which is a method to estimate regression coefficients that will maximize the likelihood of obtaining the data  for responses of the same patient to other items. This adjustment can be accomplished by propensity scores , which rIt is important to notice that although the covariates are used as predictors for the item-response, it is still impossible to infer whether this association was distorted by an association between item responses and extraneous variables rather than the association between item responses and the latent trait.i providing a positive answer to an item j is represented by:Although multiple models have been described for the regression of latent variables on predictors , we willβj represents the difficulty of item j and ui represents the trait level associated with subject i. This equation holds true in the simplest IRT model known as Rasch or one-parameter logistic (1PL). Other models – two-parameter logistic, ordinal logistic – among others – are used according to the types of response alternatives presented by each item.Where λ to represent the extent to which item j can discriminate between subjects of different trait levels, we obtain:Adding one additional parameter Finally, if we add a predictor to this equation we will haveγ is the regression coefficient for predictor x. This model allows several advantages over the two models previously described in this layer, including the absence of assumptions from summed scores as well as the summarization of all items into a single latent variable. The most frequent assumptions in IRT models are that a single construct is measured and that observations are independent, conditional on the latent variable. Different IRT models will have different assumptions about the extent to which assumptions of summed scores can be relaxed. For example, 1-Parameter. Logistic Regression models assume that each item measures the latent trait with equivalent strength.where One important practical aspect, when making use of IRT models with predictors, is to check quadrature point approximation used in the random-effects estimator. As a rule of thumb, if the coefficients do not change by more than a relative difference of 0.01%, then the choice of quadrature points does not significantly affect the outcome and the results may be confidently interpreted. Two aspects of random-effects models have the potential to make the quadrature approximation inaccurate: large group sizes and large correlations within groups .To illustrate a practical application of the previously described models, we will use data from a cohort study of patients with low-back pain to evaluate the gender-disability association. Specifically, we will evaluate whether female patients either have more severe disability or simply whether they are more likely to give positive answers to some items while having equivalent physical disability levels.Several studies have found that, compared to men, women are usually associated with higher initial disability and pain scores after low-back pain episodes ,15. HoweA description of the cohort used for this analysis is presented in detail elsewhere . BrieflyBriefly, our sample is composed by 1,633 individuals with a diagnosis of low-back pain. Most patients are females (52.3%), married (69.9%), white (83.0), and with medical insurance (68.3%).For linear and logistic regression models the data were placed in wide format, with individual variables representing patient responses to each item. For IRT models the data were presented in long compressed format Figure .When comparing the crude association between summed scores and gender, it was found that female patients had scores that were on average 1.46 (95% CI 0.73, 2.08) points higher than their male counterparts in a 0–23 scale. This association was further tested in a linear regression model Figure  controllSince the distribution of summed scores of the modified Roland Questionnaire was not normal, we used regression diagnostics using plots to determine that the relationship between predicted and observed values did not display any violations of the regression assumptions. This was confirmed by a Ramsey regression specification error test (RESET) for omitted variables (p = 0.7371) although the Breusch-Pagan / Cook-Weisberg test demonstrated a trend towards heteroscedacity (p = 0.0777).In order to further verify the robustness of this association, an ordinal logistic regression model was used with cut-points at 0–7 (low summed score), 8–15 (medium summed score), and 16–23 (high summed score). This model was considered to adequately comply with the proportionality assumption (p = 0.776). Results for the ordinal regression model demonstrated that the predicted probability of a male having low, intermediate, and high scores were progressively decreasing: 0.38, 0.33, and 0.28, respectively. This pattern was in contrast with women, where the probabilities were ascending: 0.32, 0.33, and 0.35, respectively.In summary, all results from models using summed scores point to a significant association between female gender and high disability scores. It is unclear; however, whether this association can be explained by high disability levels or simply different report patterns between men and women.As a next step, the association between individual item responses and gender was evaluated using logistic regression models stratified by propensity scores adjusting for responses to other items Figure . PropensThe analysis across propensity strata demonstrated contradictory results, with male patients being significantly associated with positive responses to items 4  and 8 ("I only stand for short periods of time because of my back problem or leg pain (sciatica)"), while female patients were significantly associated with positive responses on items 7 "), 15 , 17 ("I stay in bed most of the time because of my back or leg pain (sciatica)"), and 19 . No single item was consistently associated with gender across all propensity score strata.A new model was then built adjusting for scores pooled across strata. The results demonstrated that most items were not associated with either gender, items 4  and 8 ("I only stand for short periods of time because of my back problem or leg pain (sciatica)") being positively associated with male gender while items 7 ") and 15  being associated with female gender Figure .Since logistic regression models do not control for the latent variable one cannot test whether the association between gender and individual item responses is related to an association with disability or simply caused by women being more likely to provide a positive response to a certain item in spite of having the same degree of disability.Finally, IRT models Figure  were useTo test the hypothesis that some items might present different reporting patterns, we tested for interaction terms between each item and gender. Our results demonstrated that items 7 ", Figure A new IRT model was then calculated, but now excluding all items with differential reporting patterns. The difference in disability reporting between men and women was reduced (coefficient 0.04, log likelihood p = 0.08), indicating that gender was no longer significantly associated with disability. In fact, when the same items were excluded from the summed score, a multiple linear regression model demonstrated that the difference between female and male patients had been reduced to 0.78 points (95% CI, -0.99, 1.23) on the original 0–23 scale (p = 0.06), a reduction of 53.4% compared to the original difference.Bootstrapping methods were used in the linear regression model to verify whether the association was robust after multiple sampling procedures had been applied to the models. The results demonstrated a variation of only 13.2%; thus, indicating that these results are robust provided that the sample is representative of the study population.In conclusion, one could infer that although women still have slightly more disability than men, much of the previously reported differences using the modified Roland were inflated by the presence of items with different reporting patterns in scales measuring disability.We used three different regression models to investigate the association between gender and disability. Although summed models demonstrated a significant association between gender and disability, these models did not allow us to test whether this purported difference was related to the latent construct disability or to items presenting with differential item functioning. Analysis of the association within individual items demonstrated inconsistent associations with gender, with some items presenting a strong positive association with male gender while others had a positive association with female gender. Since these associations were made with the item response rather than the latent variable, it was impossible to verify whether these were valid representations of the construct of interest, associations with disability, or simply the effects of differential item functioning. Last, we examined the association between gender and disability measured as a latent variable. After removing items with differential item functioning, the association with gender was lessened and no longer significant. Therefore, we concluded that although a small difference between genders in relation to the disability associated with low back pain does exist, much of it is caused by differential item functioning than a true association with the disability construct.In summary, we advocate that the measurement of the association between latent variables and covariates be systematically performed using a combination of regression models to ensure that observed associations are not distorted by differential item functioning.RP: design, analysis, manuscript writing; MT: design, analysis, manuscript revision; UG: design, analysis, manuscript revision; LDH: design, manuscript revision; DOJ: design, manuscript revision; TC: data collection, design, manuscript revision.


Mycoplasma hominis vaa gene encodes a highly variable, surface antigen involved in the adhesion to host cells. We have analysed the structure of the vaa locus to elucidate the genetic basis for variation of vaa.The vaa on existing physical maps of five M. hominis isolates by pulsed field gel electrophoresis revealed that vaa is located in a genomic region containing the majority of other characterized membrane protein genes of M. hominis. Sequencing of an 11 kb region containing the vaa locus of M. hominis isolate 132 showed the presence of conserved housekeeping genes at the borders of the region, uvrA upstream and the hitABL operon downstream to vaa. Analysis of 20 M. hominis isolates revealed that the vaa upstream region was conserved whereas the downstream region was highly variable. In isolate 132 this region contained an open reading frame (ORF) encoding a putative 160 kDa membrane protein. Homologous ORFs were present in half of the isolates, whereas this ORF, termed vmp (variable membrane protein), was deleted from the locus in the remaining isolates. Compellingly, the conserved upstream region and variable downstream region of vaa correlates with the genetic structure of vaa itself which consists of a conserved 5' end and a variable 3' end containing a variable number of exchangeable sequence cassettes.Mapping of vaa locus contains a divergent genetic islet, and indicate pronounced intraspecies recombination. The high variability level of the locus indicate that it is a chromosomal 'hot spot', presumably important for sustaining diversity and a high adaptation potential of M. hominis.Our data demonstrate that the  The mycoplasmas are wall-less prokaryotes characterized by small genomes 580 – 2200 kb) and a low G+C content, generally below 30%. They are the smallest self-replicating organisms known with cell diameters normally in the range of 0.3–0.8 μm , and are200 kb anMycoplasma hominis is an opportunistic human pathogen observed as a commensal of the urogenital tract. Primarily, urogenital infections giving rise to spontaneous abortions, pelvic inflammatory disease, and acute pyelonephritis have been associated with M. hominis, but extragenital infections resulting in infant meningitis, arthritis, and septicemia have been reported [reported .M. hominis is a very heterogeneous species as measured by a pronounced antigenic variation [M. hominis surface proteins has been elucidated in some cases. The large membrane protein (lmp) gene family displays size variation by insertion/deletion of intragenic repeats of approximately 500 bp. The lmp genes are arranged in two clusters, lmp1-2 and lmp3-4 in the M. hominis genome of most analysed isolates with a distance between the clusters of more than 110 kb [lmp family is expressed in each of the M. hominis isolates tested and decrease in the number of repeats were found to correlate to the amount of spontaneous agglutination of M. hominis cells [ariation -7. The mn 110 kb . At leasis cells ,10.vaa (variable adherence-associated) gene encodes a size and phase variable M. hominis adhesin [-3–10-4. In the ON-state 8 adenines are observed in the poly-A tract, whereas 7 or 9 results in the out-of-frame OFF-state [vaa gene is present in each M. hominis isolate [vaa [vaa gene types have been observed in more than 100 analysed clinical isolates [The  adhesin -14. Phas adhesin . This isFF-state . A singl isolate . The sizate [vaa ,13. Eachate [vaa . Based ovaa and variation mechanisms in M. hominis in general, the vaa locus was characterized by mapping of the genomic position in five isolates and sequencing of a 11 kb region containing the vaa gene from isolate 132. Furthermore, the vaa locus of 20 M. hominis isolates was investigated by PCR and sequencing. In contrast to the more conserved vaa upstream region this analysis revealed that the downstream region also exhibits major variation caused by insertion/deletion and sequence variation of a large ORF encoding a putative membrane protein. Thus the vaa locus seems to constitute a 'hot spot' for variation in the M. hominis genome.To obtain a better understanding of the genomic basis for variation of vaa gene was mapped on exsisting physical and genetic maps of 5 M. hominis isolates: 132, 4195, 7488, PG21, and 93 dATP-labelled DNA fragments representing different parts of selected vaa genes . All probes hybridized to a single fragment in all digests, corresponding to the same region of the genome for all 5 isolates . The band differences observed could be explained by the presence or absence of an EcoRI site in the variable vaa gene. Using a probe comprising the cassette region of the vaa category 3 (vaa-3) gene (probe 4) band size variation was observed in the 5 isolates for both enzymes  protein encoded by hitL. This system was previously characterized in M. hominis by Henrich and coworkers [hitABL operon was located almost 5 kb downstream to the vaa gene of M. hominis 132 , encoding a hypothetical 35 kDa protein, showed high similarity to a range of hypothetical proteins of similar size in the database. All of these proteins contained a HAD hydrolase superfamily motif. The highest similarity was to a hypothetical protein of Mycoplasma pulmonis (31% identity, 54% similarity in 268 aa). No significant homologues were found for ORFs 2 to 5. ORF5 was shown to encode a hypothetical protein containing an N-terminal signal peptide with a signal peptidase II cleavage site typical of prokaryotic prolipoproteins and may thus encode a lipoprotein having a size of 29 kDa and a pI of 9.6. Interestingly, a transmembrane helix was predicted in the C-terminal part of this putative lipoprotein using the program TMHMM (membrane probability of 1 for aa 243 to 252) [Analysis of the contig harboring the mes Fig. . At the kDa Fig. . This OR to 252) .vaa revealed the presence of a large open reading frame of 4 kb, ORF6, encoding a hypothetical protein with a molecular weight of 160 kDa , and other myosin proteins in the database. In the region between ORF6 and the hitABL operon, a tRNA(His) gene was identified by database search  of Bacillus subtilis. Intriguingly, the 5' end of the gene displayed a high similarity to the orthologue from Streptococcus pneumoniae (94% identity from bp 10 to 45), even higher than to the corresponding region in Bacillus subtilis (92% identity) and Mycoplasma pneumoniae (86% identity). The transcriptional direction of ORF6, tRNA(His) and the hitABL operon was opposite that of vaa in analogy to the ORFs of the vaa upstream region . It was not possible to classify the profiles according to the vaa type or other known M. hominis groupings. Thus, despite a higly conserved organization and length of the ORFs in the vaa upstream region, there is an underlying sequence variation, presumably corresponding to the background variation level present in the M. hominis genome.To examine the variability of the ed Figs.  and 4B. vaa and hitB, respectively, were used in PCR, a pattern revealing different product sizes was observed . The isolates were divided into vmp groups based on the above PCR results according to the size of the vmp gene observed in the different sized downstream PCR products. The vmp gene having a size of 4 kb was named vmp category 1 or simply vmp-1, and the isolates  showing high homology of the stem regions to that observed for vmp-1. The stem-loop structure was located approximately 500 bp downstream of the stop codon of vmp-2 but interestingly, the homology between vmp-2 and vmp-3 extends beyond the stop codon in vmp-2. Careful analysis reveals that a poly-A tract in the 3'-end of the vmp genes has an extra  adenine in vmp-2 compared to vmp-3, which causes a premature termination of translation of the vmp-2 gene. If the extra adenine of the poly-A tract was deleted the ORF would continue for approximately 500 bp, corresponding to vmp-3, and the termination codon would be situated close the putative rho-independent transcriptional termination stem-loop structure. The remaining 10 isolates were divided into two groups based on a genetic fingerprint in the vaa-hitABL intergenic region of isolates lacking vmp  gene was different for vmp-1 and vmp-2, the latter positioned between tRNA(His) and hitABL, whereas the more similar vmp-2 and vmp-3 genes were positioned identically  sequence box between the insertion site in isolate PG21 and the stem region of the stem-loops of vmp-2 and vmp-3. Furthermore, a 165 bp deletion  intergenic region of isolate 7488 compared to isolate PG21. This deletion was located immediately downstream to the region deleted in isolate 93. In contrast, analysis of the vmp-1 insertion site did not show insertion at the stem-loop and poly-T structures when compared to the vaa-hitABL intergenic regions of isolates PG21 and 93. Sequence regions of 60 bp downstream to the stem-loop structure and 90 bp upstream to the poly-T tract which did not show any homology to the vaa-hitABL intergenic region of isolate PG21 were observed at the borders of the vmp-1 gene. Interestingly, comparison of the insertion site of the vmp-1 region including the 60 bp and 90 bp bordering sequences with the vaa-hitABL intergenic region of isolate PG21 revealed that insertion resulted in a deletion in the intergenic sequence corresponding to the 300 bp deletion . Half of this region was repeated once in Vmp-3 Fig. . The N-tSequence analysis revealed that the deduced Vmp proteins have a predominantly alpha-helical structure, and a coiled-coil region extending throughout almost the entire length of the proteins was identified Fig.  and 6C. vmp-1 gene of these isolates. These data thus demonstrate that Vmp is expressed in M. hominis. Furthermore, the antibody reacted with a 100 kDa protein in all isolates except PG21. This is presumably a protein that has been shown previously to bind Ig-molecules unspecifically, and is not present in PG21 [vmp gene, and isolates 4195 and 7488 having a Vmp-2 and Vmp-3 type, respectively, did not react with the antibody, as expected from the low homology of the C-terminal region  gene, and the lack of correlation of vmp type and absence of vmp to vaa type suggests frequent recombination in this locus  or genetic islands (>10 kb), are variable sites when comparing genomes of different isolates of a given species. Often, genetic islets/islands carry pathogenesis factors and are specific for virulent clones of the species. Such factors include adhesins, toxins and restriction/modification systems. Frequently, the genetic elements are inserted into tRNA genes, show a GC content diverging from the surrounding regions and are flanked by repeated elements [vmp gene was located on either side of the tRNA(His) gene in isolates 132 and 7488, respectively, the GC content of vmp was similar to the remaining part of the vaa locus analyzed and the M. hominis genome in general (28%) and despite a thorough analysis, no obvious flanking structures such as direct or inverted repeats were observed which could indicate a site specific mechanism of insertion. Thus, the vmp gene may be mycoplasma specific and insertion/deletion of vmp at the vaa locus seems mediated by homologous recombination.The location of elements . Althougvmp fragments by PCR from four out of five isolates categorized as having a vmp-1 gene type and likewise for four out of five isolates having a vmp-2 or vmp-3 gene type. The pronounced heterogeneity observed between the vmp genes could explain the missing reaction of the remaining two isolates as being caused by sequence variation of the individual gene types as observed for a number of other M. hominis membrane protein genes, but it is also possible that additional vmp gene types exists.It was possible to amplify Mycoplasma pneumoniae. This protein is cytoplasmic, part of the primitive cytoskeleton of this mycoplasma and truncation of the gene resulted in loss of cytadherence [M. hominis in contrast to the cytoplasmic HMW2.The size and predicted structure of Vmp is interesting. The coiled-coil motif extending through most of the protein, is a highly versatile motif involved in protein-protein interactions . This modherence . HMW2 isdherence . In contM. hominis.The identification of a novel putative membrane protein displaying sequence variation is intriguing, and furthermore the remarkable size and structure displayed by the Vmp protein is interesting and should prompt investigations on the biological function of this protein in vaa locus of M. hominis contains a divergent genetic islet encoding a large, putative membrane protein called Variable membrane protein (Vmp). This genetic islet is only present in the locus of half of the 20 islolates tested, and three distinct, homologous Vmp types were observed. The composition of the locus was analysed and it was found that the vaa gene has a conserved upstream region and a highly variable downstream region, which contains the genetic islet. This locus organization corresponds to the organization of the vaa gene itself having a conserved 5' end and a variable 3' end. Thus, the mechanism underlying variation of the vaa gene seems to be intraspecies recombination exchanging variable regions of vaa and downstream regions of vaa, giving rise to a variable and dynamic 'hot spot' in the M. hominis genome.We have demonstrated that in some isolates, the M. hominis isolates were analysed (PG21, 4195, 132, 5941, 1621, 7488, 93, 3105, 1572B, 2032B, 2347B, 7808, 7357, 4712, V2785, DC63, SC4, P2, 183, and P71). The M. hominis isolates were cultivated in BEa medium (heart infusion broth (Difco), 2.2% (w/v); horse serum, 15% (v/v); fresh yeast extract, 1.9% (w/v); benzylpenicillin, 40 IU ml-1; L-arginine, 0.23% (w/v); phenol red 0.0023% (w/v)). The pH of the medium was adjusted to 7.2 and the medium was sterilized by filtration [M. hominis isolates were harvested by centrifugation at 15,000 rpm for 45 min at culture volumes greater than 1.5 ml or at 20,000 rpm for 15 min for culture volumes smaller than 1.5 ml. E. coli OneShot competent cells and the pCRII plasmid vectors were used for TA-cloning (Invitrogen).Twenty ltration . The M. M. hominis isolates PG21, 4195, 132, 93 and 7488. The M. hominis isolates were grown in BEa medium to log phase and harvested. The cell pellets were washed and resuspended in PBS buffer (20 mM sodium phosphate, 250 mM NaCl, pH 7.4). Melted NA agarose (Amersham Pharmacia Biotech) was mixed with the cell suspension in a plastic mold on ice. The agaroseblocks hereby formed were incubated overnight with 1 mg/ml Proteinase K (Roche) in lysis buffer (1% Sarcosyl, 0.5 M EDTA, 10 mM Tris HCl, pH 9.5). Subsequently, each block was washed twice in lysis buffer followed by one wash in TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and cut into eight blocks of identical size. The blocks were digested overnight with 40 units of one of five restriction enzymes  and 12 mg of BSA. Subsequently, the blocks were inserted into the slots of 1% NA agarose gels and the holes sealed with melted NA agarose. HindIII digested λ DNA and a λ DNA ladder (FMC) was used as molecular weight markers. The gels were used for PFGE using the CHEF-DRII separation system (Bio-Rad).PFGE was performed on genomic DNA from the five M. hominis isolates was isolated using the method described in [M. hominis cells were harvested and subsequently lysed on ice in a buffer containing 0.7% (w/v) N-laurylsarcocine, 10 mg RNase ml-1 (Sigma), 20 mM Tris pH 7.5 and 20 mM EDTA. Proteinase K (150 mg ml-1) was added and the cell lysate was incubated at 55°C for 2 hrs and 37°C for 1–2 hrs followed by phenol, phenol/chloroform and chloroform extractions [-1) treatment of harvested M. hominis at 55°C for 1 h. After the incubation the solution was heated to 100°C for 5 min to inactivate the enzyme. Plasmids from transformed E. coli were prepared as described in [DNA from the ribed in . Brieflyractions . DNA preribed in , for seqvaa types with sizes of 1220 bp (probe 1), 600 bp (probe 2), 800 bp (probe 3), and 660 bp (probe 4) were used for TA-cloning, performed according to manufacturers instructions (Invitrogen) [vaa-1 gene from M. hominis 7808, including the three cassettes III, IV and V [vaa-1 gene of M. hominis PG21 and probe 3 contained most of the vaa-3 gene of M. hominis V2785 including cassettes V and VII. TA-cloned PCR fragments or linear PCR fragments were used as DNA probes and labeled with radioactive (α-32P)dATP by nick-translation, performed as follows. 0.5–1 μg DNA was mixed with 1 × nick-translation buffer (50 mM Tris HCL (pH 7.2), 10 mM MgSO4, 0.1 mM DTT, 50 mg BSA, 60 mM of dTTP, dCTP, and dGTP respectively, 5 units of DNA polymerase I (Gibco), 0.5 ng DNase I (Roche), 20 mCi (α-32P)dATP (Du Pont) and ddH2O up to 50 μl. The reaction was incubated at 14–16°C for 1 h. Incorporation of radioactive nucleotides was verified by TLC and terminated by addition of TE-buffer with 0.5 M EDTA. The radioactive probes were denatured by heating to 100°C for 5 min and hybridization performed in 2 × SSC (1 × SSC is 0.15 M NaCl and 0.015 M sodium citrate), 0,5% SDS, 100 mg/ml yeast RNA, 5 × Denhardts solution (0.1% Ficoll, 0.1% BSA, 0.1% polyvinyl pyrollidone (Serva)) at 60°C in a hybridization oven. The membranes were washed in 6 × SSC and 0.5% SDS. The membranes were placed in sealed plastic bags and exposures of X-ray films were performed at room temperature or at -20°C.PCR-products derived from different itrogen) . Probe 1IV and V . Probe 2HindIII or EcoRI and separated on 0.7% agarose gels. The gels were stained with ethidium bromide and photographed under UV irradiation. Preceding the alkaline denaturation, partial hydrolysis of the DNA in the PFGE gels was performed by soaking in a 0.25 M HCl solution to enhance the transfer of large DNA fragments. DNA transfer to Hybond-N membranes (Amersham Biosciences) was carried out as described in [Genomic DNA samples of the isolates PG21, 4195, 132, 93 and 7488 were cleaved with either ribed in .vmp-2/3 PCR product where Taq polymerase was used (PE Biosystems). Custom oligonucleotide primers were purchased from DNA Technology (Aarhus, Denmark). PCR products were purified using the Wizard kit (Promega) according to manufacturers instructions. PCR conditions used for inverse PCR and amplification of downstream and upstream regions of the 20 isolates were as follows: 2 min at 92°C, 10 cycles of 10 s at 92°C, 30 s at 55°C, 8 min at 68°C, 20 cycles of 10 s at 92°C, 30 s at 55°C, 8 min at 68°C with 5 s added to the elongation time pr. cycle. Finally, an extension step of 7 min at 68°C was performed. The primers used for amplification of the 5.5 kb upstream product were F1 (CAGTACATGTTAATCCCAGAA GTATAGTTGG) and R1 (GCTGGATAATCGCCGTATGAACCTGC). The R1 primer was also used for amplification of the 4 kb and 0.6 kb PCR products in combination with the primers F2 (GGATCTTCTTTGTGGTCTTCC) and F3 (GGGATAGTTAGTAAAG TTGGAATAGCC), respectively. For amplification of the downstream region in the 20 isolates, the primers F4 (GCAGGTTCATACGGCGATTATCCAGC) and R4 (GCCACTTGCGGTTCTTCC) were used. For the amplification of the 0.6 kb vmp-1 PCR product, the primers F6 (CCACTGATACGTGATTTAAAAAGAAAAG) and R3 (GGTATTGTTTCTTTATCTAAGATGTTTTCAAATTC) were used with the following PCR conditions: 4 min at 94°C, 30 cycles of 15 s at 94°C, 30 s at 50°C, 1 min at 72°C and a final extension of 5 min at 72°C. For amplification of the 1.2 kb vmp-2/3 PCR product, similar conditions were used with an annealing temperature of 57°C and an elongation time of 2 min, and the primers were F5 (GAACAATTAAAAACATTAATTGGCTTAA GTGATG) and R2 (GTTTTATCTACATTGTTTTCGGATAAGG).PCR was performed using the Expand™ High Fidelity PCR System from Roche according to the manufacturer's instructions, except for the amplification of the 1.2 kb AluI and AseI (New England Biolabs) according to manufacturers instructions and analysed on 1 × TBE/2% agarose gels.The 5.5 kb upstream PCR products from the 20 analysed isolates were subjected to restriction endonuclease analysis employing the enzymes Sequencing reactions were carried out bidirectionally using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) on purified plasmid DNA (TA-cloned PCR products) or directly on the purified PCR products according to the instructions supplied by the manufacturer. Sequencing was performed on an ABI PRISM 377 DNA Sequencer from Perkin Elmer.vmp-1 from isolate 132 encoding aa 1281 to 1404 of the Vmp-1 protein. Cloning and expression of the construct was performed using the pET-30 Ek/LIC vector according to manufacturers instructions (Novagen, Madison, USA). The His-tagged fusion protein was purified using a nickel chelated column (High Trap Sepharose, Amersham Pharmacia Biotech) under denaturing conditions as previously described [Oligonucleotide primers (DNA technology, Aarhus, Denmark) were designed in order to amplify by PCR, the region of escribed . Sera co, were used to predict signal sequences and transmembrane helices, respectively [[Computer analysis of the obtained DNA sequences was performed using the Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisc., sequence analysis software package . Data baectively ,24. Enerctively [.The DNA sequences obtained in this study were deposited to the EMBL database under the following accession numbers: AJ416752, AJ545046 AJ629113, AJ629114 and AJ629115.The individual parts of the work presented in the paper were conducted as follows: The ideas and designs of the experiments were developed by all the authors. Pulsed field gel electrophoresis and Southern blottings were performed by JE and TB. PCR, sequencing, and sequence analysis were performed by TB and AB. The fusion protein, polyclonal antisera and immunoblotting were made by TB. The manuscript was primarily written by TB and discussed with and approved by all authors.Multiple sequence alignment. Alignment of the three Vmp types and Lmp1 and Lmp3 from type strain PG21 using ClustalW.Click here for file


Helicobacter pylori, occurring throughout the world and causing gastroduodenal diseases, is one of the most common chronic bacterial agents in humans. The purpose of this study was to measure the general practitioners' (GPs) knowledge and practices pertaining to H. pylori infection.H. pylori.A cross-sectional type questionnaire survey was conducted in all of 19 primary health care centres (PHCC) in Samsun, Turkey, between November 1 and December 31, 2003. The questionnaire was sent to 124 GPs and 109 (87.9 %) of those filled in. They were requested to answer the questions on the knowledge, sources of medical information, diagnostic tests and treatment to H. pylori, being cited by 86 (78.9%) of GPs. Ninety-two (84.4%) of the GPs reported having used one or more tests and 17 (15.6%) never used any test for the diagnosis of H. pylori infection. Only 9.8% had used stool antigen test for diagnosis. GPs reported that they would prescribe symptomatic treatment without ordering diagnostic tests for 29 (26.6%). 54.1% of the GPs explain that they sent patients with H. pylori infection to a specialist, and most used a triple drug regimen containing a PPI. Treatment duration varies between 7 to 28 days. 80.7 of the GPs treat patients for 14 days.Medical journals were the most frequently used source of information on H. pylori infection during post-graduation period should be improved in PHCCs.GPs may not have enough knowledge about the importance of stool antigen test or possibility of usage of this test. GPs have not sufficient knowledge about the difference between symptomatic and asymptomatic individuals. It is thought that GPs preferred to treat the patients with suspected ulcer empirically or to send them to a specialist because of the limited diagnostic conditions. The efforts to educate the GPs about the algorithms regarding the management of  Helicobacter pylori, occurring throughout the world and causing gastroduodenal diseases, is one of the most common chronic bacterial agents in humans [H. pylori is increasing, most ulcers are related to H. pylori infection [H. pylori infection have been promoted in order to improve early detection and treatment of ulcers in dyspeptic patients [n humans . Althougnfection ,3. For mpatients ,5.H. pylori infection in patients with chronic gastritis and peptic ulcer disease in 1983 has fundamentally changed concept of the etiologic, pathogenesis and management of upper gastrointestinal (UGI) diseases [H. pylori related information and the development and publication of international, regional and national guidelines [H. pylori infection and the pathogenesis, diagnosis and treatment of UGI diseases [H. pylori infection and treat if positive, and when to refer patients to a specialist [The successful isolation of diseases . This haidelines . Subsequdiseases . The majH. pylori infection.It is thought that too many patients with dyspeptic symptoms apply to GPs in Turkey. This study was performed a survey of GPs' to assess their knowledge and practices pertaining to H. pylori knowledge section contained 6 questions. The items assessed respondents' knowledge of diagnosis of infection, case selection for treatment and treatment options in H. pylori. Participating GPs were asked to offer the type(s) of diagnostic tests such as ELISA, histology, biopsy urease test (BUT), urea breath test (UBT) or culture of biopsy specimen.A cross-sectional study was conducted in all of 19 primary health care centres (PHCC) in Samsun, Turkey, between November 1 and December 31, 2003. The questionnaire was sent to all GPs (n = 124). 109 of 124 (87.9 %) GPs from different PHCCs completed the survey. The material used was adapted from the questionnaire devised by Sharma et al. [H. pylori and also treat the infection when the test results were positive for H. pylori.A list of 7 different clinical presentations was given. It was asked whether the respondents would offer testing for H. pylori infection among the list of four drug combination regimens included proton pump inhibitor (PPI)-based triple therapies. [H. pylori infection before or after the survey in this study period. Data were given as mean ± standard deviation (SD) and percentage.The respondents were asked to select a regimen for the management of erapies. ,11,12. GThe mean age and working year of the GPs was 31.7 ± 5.4 and 7.2 ± 5.0 years, respectively; 59 (54.1%) of the GPs were women.H. pylori, being cited by 86 (78.9%) of GPs. Pharmaceutical company-sponsored symposia (70.6%), textbooks (64.2%), conferences (20.2%) and on-line sites (6.4%) were the other major source of information used by the GPs. These numbers add up to more than 100% because the GPs had been checked more than one item.Medical journals were the most frequently used source of information on H. pylori infection. Of those, 44.1% had used UBT, 34.5% had used BUT, 23.8 % had used ELISA and 9.8% had used the stool antigen test. The practitioners included in the survey had not equally access to all diagnostic tests mentioned in the study.Ninety-two (84.4%) of the GPs reported having used one or more tests and 17 (15.6%) never used any test for the diagnosis of H. pylori infection in the 9 different clinical situations and the proportions of those who would offer treatment based on a positive test result, were summarized in Table-H. pylori infection to a specialist.The proportions of GPs who would test patients for Treatment regimens of choice are listed in Table-H. pylori detection methods, including ELISA and the UBT, has enabled GPs to diagnose and treat H. pylori infection. The inadequate treatment of peptic ulcer disease results in therapy failures, high recurrence rates, the emergence of resistant bacterial strains, and increased health care costs, therefore clinical application of current knowledge is crucial [UGI symptoms are common reasons for patients to visit GPs. In recent years, the development of non-invasive  crucial ,14.H. pylori infection [H. pylori infection same as Huang J et al.'s study [There are several tests used for diagnosis of nfection ,13,15,16's study . UBT is 's study .H. pylori infection are recommended following resection of early gastric cancer and for low-grade gastric MALT lymphoma. Retesting after treatment may be prudent for patients with bleeding or otherwise complicated peptic ulcer disease [It is worrisome that considerably fewer perceived a need for testing and subsequent treatment in patients with a new diagnosis and past history of duodenal ulcer Table . Both th disease .H. pylori infection, 64.1% of GPs reported that they would offer testing for H. pylori infection and 31.5% of them reported that they would treat H. pylori infection based on a positive test result in asymptomatic individuals. These findings suggest that GPs have not sufficient knowledge about the difference between symptomatic and asymptomatic individuals. On the other hand, in any person testing positive for the infection, treatment may be offered after a full discussion about its potential risks and benefits [Although it is not recommended to test the asymptomatic individuals for benefits .H. pylori therapy was almost never recommended for suspected ulcer disease without the prior use of diagnostic tests [H. pylori infection. 54.1% of the GPs, whether ordering diagnostic tests or not, explain that they send patients with suspected or diagnosed H. pylori infection to a specialist. In the light of these findings, it is thought that GPs preferred to treat the patients with suspected ulcer, empirically or to send them to a specialist because of the limited diagnostic conditions, the lack of rapidly diagnostic tests at PHCCs, or they thought that they should be treated by a specialist.Anti-ic tests ,18. In tH. pylori peptic ulcers are treated with drugs that kill the bacteria, reduce stomach acid, and protect the stomach lining. Antibiotics are used to kill the bacteria. Two types of acid-suppressing drugs might be used: H2 blockers and PPI. H2 blockers and PPI have been prescribed alone for years as a treatment for ulcers. When used alone, these drugs do not eradicate H. pylori and, therefore, do not cure H. pylori-related ulcers. Bismuth subsalicylate, a component of Pepto-Bismol, is used to protect the stomach lining from acid. It also kills H. pylori [. pylori ,7,12,19.H. pylori infection but 15.6% never test. Urea breath test is a commonly used investigative tool for H. pylori infection. Triple therapy consisting of a proton pump inhibitor, clarithromycin and amoxicillin is the most commonly used treatment combination for H. pylori infection.The highest eradication rates are achieved with the following regimens: a PPI, clarithromycin, and either amoxicillin or metronidazole for 2 week; ranitidine bismuth citrate, clarithromycin, and either amoxicillin, metronidazole, or tetracycline for 2 week; a PPI, bismuth, metronidazole, and tetracycline for 1 to 2 week ,11,12,20H. pylori infection among GPs. Our data suggested that pharmaceutical company-sponsored symposia were used very frequently among GPs.It was found that most of the information was being obtained in traditional teaching formats such as medical journals, pharmaceutical company-sponsored symposia, textbooks and conferences. Other studies ,13,17) c,17 c13,1H. pylori infection [Patients with dyspeptic complaints are mostly managed in primary care. The most prescriptions for dyspepsia are empirical without testing due to limitations of diagnostic facilities around the world . Similarnfection ,17.H. pylori is not adequate. The choice of optimal therapeutic decision depends on the appropriate definition of the disease. In order to provide accurate diagnosis and treatment of H. pylori infection, it's suggested that efforts to educate the GPs about the algorithms regarding the management of H. pylori infection during post-graduation period should be improved in PHCCs.The diagnosis of and treatment of peptic ulcer disease related to The author(s) declare that they have no competing interests.SC participated in the design and coordination of the study; ATS provided drafted the manuscript and performed the statistical analysis. YP drafted the questionnaire and participated in study design and coordination. HL conceived the study, participated in its design and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: