However, its impact on soft drusen, GA, or neovascular AMD—or the relationship between them—is unclear.Age-related macular degeneration (AMD) is the most common cause of irreversible visual impairment in the developed world. The two forms of advanced AMD, geographic atrophy and neovascular AMD, represent different pathological processes in the macula that lead to loss of central vision. Soft drusen, characterized by deposits in the macula without visual loss, are considered to be a precursor of advanced AMD. Recently, it has been proposed that a common missense variant, Y402H, in the Complement Factor H CFH Y402H variant shows significant association to advanced AMD, with odds ratio of 2.39 in Icelandic patients (p = 5.9 × 10−12) and odds ratio of 2.14 in US patients from Utah (p = 2.0 × 10−9) with advanced AMD. Furthermore, we show that the Y402H variant confers similar risk of soft drusen and both forms of advanced AMD (GA or neovascular AMD).We genotyped 581 Icelandic patients with advanced AMD , and 435 with early AMD (of whom 220 had soft drusen). A second cohort of 431 US patients from Utah, 322 with advanced AMD (244 neovascular AMD and 78 GA) and 109 early-AMD cases with soft drusen, were analyzed. We confirmed that the CFH is a major risk factor of soft drusen, and additional genetic factors and/or environmental factors may be required for progression to advanced AMD.Soft drusen occur prior to progression to advanced AMD and represent a histological feature shared by neovascular AMD and GA. Our results suggest that  A common missense variant, Y402H, in the Complement Factor H gene is associated strongly with soft drusen, a precursor of advanced age-related macular degeneration Age-related macular degeneration (AMD) includes a wide range of phenotypes. Early AMD is characterized mainly by the presence of soft drusen in the macula without visual loss, while advanced AMD is characterized by geographic atrophy (GA or dry AMD) and neovascular AMD (wet AMD) with visual loss. Despite the rising prevalence of AMD as a result of increasing life expectancy, its underlying pathogenesis is poorly understood and there are limited treatment options available. Nutritional supplements and antagonists of vascular endothelial growth factor have been reported to decrease visual loss in neovascular AMD somewhat . Drusen CFH and AMD in the United States [Several genome-wide linkage scans for AMD including ours (unpublished data) have found suggestive linkage on chromosome 1q –15. The d States ,16–19. Td States . CFH prod States .However, the question of how the Y402H allele contributes to the different subtypes of AMD has not been properly addressed, owing either to insufficient clinical information or to sample size, as neovascular AMD dominates the patient cohorts in most previously reported studies ,16–19.CFH and AMD we performed genotype–phenotype correlations on different clinical subtypes of early and advanced AMD in US and European populations.In order to investigate the association between This study was approved by the Data Protection Authority of Iceland and the National Bioethics Committee of Iceland, and the Institutional Review Board of the University of Utah. All participants signed written informed consent prior to participation in the study. All personal identifiers associated with blood samples, medical information, and genealogy were encrypted. For samples from Iceland, encryption was carried out by the Data Protection Authority, using a third-party encryption system . The IceAll participants in both populations went through a standard examination protocol and visual-acuity measurements. Slitlamp biomicroscopy of the fundi using 90-diopter lenses was performed. A pair of stereoscopic color fundus photographs (50°) were taken, centered on the fovea using a Topcon fundus camera  by a trained ophthalmic photographer. Grading was carried out using a standard grid classification suggested by the International Age-Related Maculopathy Epidemiological Study Group for age-related maculopathy and AMD . All abnThe Icelandic cohort that was genotyped included 581 patients with advanced AMD and 435 patients with early AMD, and allele frequencies were compared to that of either 891 population controls or 171 longevous healthy controls . The UtaCFH Y402H variant (rs1061170), we used Fisher's exact test to calculate one-sided p-values for the at-risk allele. As the patient cohort was recruited as families for a linkage analysis, we also repeated the test for association, correcting for the relatedness of the patients by extending a variance-adjustment procedure described previously [p-values are presented for comparison. We calculate the odds ratio (OR) of the frequency of the at-risk allele as OR = p/(1−p)/s/(1−s), where p and s are the frequency of the at-risk allele in the patients and in the controls, respectively. In the case of population controls and assuming the multiplicative model, in which the risks of the two alleles of the single-nucleotide polymorphism a person carries multiply [, this corresponds to an estimate of the relative risk of the mutation compared to the wild-type. Specifically, with population controls and the multiplicative model, it can be shown through Bayes' Rule that the OR, as defined above, corresponds to Risk(CT)/Risk(TT) = Risk(CC)/Risk(CT), where C is the mutated allele and T the wild-type, and Risk is the probability of disease given the genotype.For the single-marker association of the eviously  for sib-multiply ,26, thisOn the basis of the frequency of the at-risk allele and the relative risk, we calculate the population-attributable risk or the reduction in the number of disease cases if the at-risk allele was removed from the population, again assuming the multiplicative model. Confidence intervals of relative risks and ORs were based on the variance-adjusted tests for association, assuming a log-normal distribution. To avoid confusion and to be consistent, we report the results as OR when using healthy controls and as relative risk when using population controls.In agreement with previous reports –19, the CFH variant contributes equally to GA and neovascular AMD in our European and US cohorts.The comparable association to neovascular AMD and GA was replicated in the Utah cohort, giving ORs of 2.17 and 2.05, respectively . Therefop = 0.01) to hard drusen but with a lower OR (1.57) . This CFH variant also confers increased risk although this is not significant when comparing hard drusen to controls in the Utah cohort  . A signihort see .CFH Y402H allele in four ethnically diverse populations from the International HapMap project [CFH Y402H in Caucasians and Africans were similar, 39% versus 30.7%, while they were much lower in Asians—8.1% in the Japanese and 6.8% in the Chinese.We also typed the  project ; CaucasiCFH Y402H allele confers significant risk to neovascular AMD, GA, and soft drusen in early AMD in US and European Caucasian populations. The effect of the CFH Y402H allele on soft drusen in early AMD is similar to its effect on the advanced forms of AMD, neovascular AMD, and GA. Advanced AMD is considered to be one disease with two different end-stage lesions, i.e., choroidal neovasculariztion and GA. Leaky choroidal neovascular blood vessels between Bruch's membrane and RPE are seen typically in neovascular AMD, while GA is characterized by RPE atrophy and overlying photoreceptor loss. Soft drusen located between the RPE and Bruch's membrane are usually precursors of both forms of advanced AMD. The 5-y incidence of soft drusen in the Reykjavik Eye Study, a population-based epidemiological study of individuals aged 50 y and older [.It is an open question whether the high GA/neovascular AMD ratio in Iceland is due to genetic or environmental factors that increase the risk of GA or decrease the risk of neovascular AMD. Consumption of fresh fish and fishliver oil with omega-3 polyunsaturated fatty acids among Icelanders is among the highest in the world [We have shown that the nd older ,29, was nd older , and thend older . Neovascnd older ,29.It ishe world . Intereshe world  reportedCFH as an important AMD gene, contributing to the common form of AMD and its confirmation by several groups, is a major advance towards understanding the genetic risk and pathogenesis of AMD. It is apparent that this single common variant confers similar risk in all of the US populations tested and, furthermore, our result for the association of the CFH variant to advanced AMD in a European population is comparable to that in the US. However, the previously reported studies had not adequately addressed the effect of the CFH variant on GA or early AMD. We therefore tested the reported variant in the CFH region for association to the subtypes of AMD in both of our cohorts.The discovery of CFH is thought to aid in keeping the complement pathway of the innate immune system in check. It is tempting to postulate that a hypothetical lower activity of complement H protein with the histidine variant may lead to increased inflammation that would contribute to the neovascular form of AMD as suggested before [CFH variant contributes equally to GA and to neovascular AMD, would tend to refute the first hypothesis and lend support to the second.Functionally, d before . Alternad before . Our resCFH variant on risk of soft drusen without advanced AMD. Interestingly, this CFH variant confers risk of soft drusen with similar OR (2.52) as with both forms of advanced AMD, even before the fundoscopic findings and visual loss fulfil the criteria for advanced AMD in two independent cohorts. Conversely, there is little or no impact of the CFH variant on other features such as pigmentary changes and hard drusen. Therefore, it appears that the Y402H variant in CFH contributes to the increased risk of advanced AMD largely or entirely through its impact on the development of soft drusen as a precursor of advanced AMD.Given that the protein component of soft drusen includes members of the complement system (including Complement H), we tested the effect of the CFH and its binding partner, complement 3b [CFH Y402H causes soft-drusen formation, but it is not sufficient for progression to advanced AMD. To substantiate this hypothesis further, it will be interesting to correlate the prevalence of soft drusen with advanced AMD in Asian populations. Our analysis of the CFH Y402H variant demonstrated that its frequency is less in Asian populations: 0.08 in the Japanese samples and 0.07 in the Chinese. Indeed, AMD in Asians is considered to be infrequent, but careful genotype–phenotype correlation studies are needed in non-Caucasian populations.This observation may not be surprising given that soft drusen is comprised, in part, of complement proteins including ement 3b . Howeverement 3b . The preement 3b . Interesement 3b ,35. The CFH variant is a risk factor for soft drusen, but not for hard drusen or pigmentary changes, per se. Therefore, there may be other genes that influence the appearance of hard drusen and pigmentary changes. In addition, the difficulty in explaining the difference in the ratio of the prevalences in neovascular AMD and GA across populations through the CFH variant alone supports the notion that additional genetic or environmental factors contribute to the pathogenesis. It is likely that there are other important genes, yet to be found, that contribute to the risk of advanced AMD, particularly among those who already have soft drusen.Significant genetic influence in early AMD has been demonstrated in a classical twin study comparing concordance of 226 monozygotic and 280 dizygotic twin pairs, with soft drusen and multiple hard drusen showing strong genetic influences with heritability of 57% and 81%, respectively . This ishttp://www.ncbi.nlm.nih.gov/entrez) accession number for the gene discussed in this paper, CFH, is ID 3075. The OMIM (http://www.ncbi.nlm.nih.gov/entrez) identification number for AMD, ARMD1, is 603075; and for the CFH gene is 134370.The LocusLink (The commonest cause of poor eyesight in later life in the developed world is known as age-related macular degeneration (AMD). The macula is the central part of the retina (the film-like membrane at the back of the eye) which is the most sensitive and important for sharp central vision. An early sign of AMD is what are called “drusen”—deposits of protein, fat, and cells—which doctors can see in the back of the eye. There are two types of advanced AMD: so-called “wet” or neovascular AMD (neovascular means “new vessel”) and “dry” or geographic atrophy AMD (atrophy means to waste away). Wet AMD occurs when abnormal, fragile blood vessels grow under the macula behind the retina. These blood vessels often leak blood and fluid, which lift the macula. Dry AMD occurs as the light-sensitive cells in the macula (the rods and cones) break down.(CFH), which codes for a protein that is involved in keeping one part of the immune system in check. A variant of CFH has been previously shown to be present more frequently in people with advanced AMD compared to normal controls. These investigators wanted to go further, to find out whether this variant was more linked to the wet or to the dry type of AMD and to early AMD.Although this disease is common, little is understood about why it occurs, and current treatments have limited efficacy. Previous studies have suggested that a gene in a particular part of Chromosome 1 is linked to the chance of getting AMD. The responsible gene is Complement Factor H CFH in two groups of patients with various types of AMD, 1,118 from Iceland and 431 from Utah, and compared the results with people without AMD from the same ethnic groups and age. As had been shown before, they found that one variant of this gene occurred more frequently in the wet form of AMD. However, they report two new observations. First, the variant of CFH also confers risk for the dry form of AMD and second, the variant confers similar risk to drusen in the early form of AMD.They looked at the variant of It appears that this gene variant is important early on in the development of AMD—which makes sense as the protein for which this gene codes is involved in keeping the immune system under control. The particular variant found here may not be as efficient as the normal one—that is, it makes it more likely that inflammation will develop in the eye. These findings do not have any immediate implications for treatment, but they suggest that there are other genes that cause the severe forms of AMD with blindness.Here are several Web sites with information on macular degeneration.MedlinePlus:http://www.nlm.nih.gov/medlineplus/ency/article/001000.htmNational Institutes of Health Senior Health:http://nihseniorhealth.gov/agerelatedmaculardegeneration/toc.htmlNational Eye Institute:http://www.nei.nih.gov/health/maculardegen/armd_facts.aspPrevent Blindness America:http://www.preventblindness.org/eye_problems/amdFAQ.htmlFoundation Fighting Blindness:http://www.blindness.org/MacularDegeneration/


Congenital abnormalities of the kidney and urinary tract are frequently observed in children and represent a significant cause of morbidity and mortality. These conditions are phenotypically variable, often affecting several segments of the urinary tract simultaneously, making clinical classification and diagnosis difficult. Renal agenesis/hypoplasia and dysplasia account for a significant portion of these anomalies, and a genetic contribution to its cause is being increasingly recognized. Nevertheless, overlap between diseases and challenges in clinical diagnosis complicate studies attempting to discover new genes underlying this anomaly. Most of the insights in kidney development derive from studies in mouse models or from rare, syndromic forms of human developmental disorders of the kidney and urinary tract. The genes implicated have been shown to regulate the reciprocal induction between the ureteric bud and the metanephric mesenchyme. Strategies to find genes causing renal agenesis/hypoplasia and dysplasia vary depending on the characteristics of the study population available. The approaches range from candidate gene association or resequencing studies to traditional linkage studies, using outbred pedigrees or genetic isolates, to search for structural variation in the genome. Each of these strategies has advantages and pitfalls and some have led to significant discoveries in human disease. However, renal agenesis/hypoplasia and dysplasia still represents a challenge, both for the clinicians who attempt a precise diagnosis and for the geneticist who tries to unravel the genetic basis, and a better classification requires molecular definition to be retrospectively improved. The goal appears to be feasible with the large multicentric collaborative groups that share the same objectives and resources. Human urinary tract abnormalities are phenotypically variable and may affect several segments simultaneously, often aggregating to form complex phenotypes. Hence, clinical classification and diagnosis may be difficult. As a consequence of the overlap between anatomical defects, many investigators have opted to group renal and urologic malformations under the single label of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) [PAX2 gene cause the renal-coloboma syndrome, but the clinical features of the trait vary significantly between affected individuals, ranging from renal agenesis/hypoplasia to vesicoureteral reflux (VUR) and secondary obstruction [EYA1 and PAX2 mutations both can cause the development of hypoplastic kidneys [Congenital abnormalities of the kidney and urinary tract are frequently observed in the first year of life, when they collectively represent a significant cause of morbidity  and mort (CAKUT) . This brtruction . Convers kidneys . Hence, Bilateral renal agenesis is a rare and fatal event, usually associated with severe oligohydramnios, which produces a characteristic clinical pattern with facial compression and pulmonary hypoplasia (Potter syndrome). An estimate of the incidence of bilateral agenesis is 0.1/1000 births. Unilateral renal agenesis is more common, although the frequency is difficult to estimate, as it is usually clinically silent and is commonly detected as a chance observation by autopsy or by prenatal ultrasound .Strictly speaking, renal hypoplasia is defined as a small kidney, which contains intact nephrons that are reduced in number, whereas a dysplastic kidney contains disorganized elements and maldifferentiated tissue. Noninvasive imaging studies such as ultrasounds and dimercaptosuccinic acid (DMSA) scan offer limited information to help distinguish a hypoplastic kidney from a dysplastic one. Unequivocal distinction between these two entities therefore depends on histological examination of renal tissue obtained from kidney biopsy or surgical nephrectomy, which are rarely performed. A further confounding factor is the reduction of kidney size due to chronic injury and scarring from VUR. Most of the time, a DMSA scan helps differentiate primary hypoplasia or dysplasia from small kidneys secondary to VUR. However, a DMSA scan has a low negative predictive value in distinguishing primary hypoplasia or dysplasia from a secondary reduction in kidney size from VUR when scars or areas of negative isotope uptake are present. In practice, the diagnosis of primary renal hypoplasia is favored when the following criteria are satisfied: (a) a reduction of renal size by 2 standard deviations (SDS) from the mean size for the age, (b) exclusion of renal scarring by DMSA scan, and (c) a presence of compensatory hypertrophy of the contralateral kidney. In all cases, the exclusion of renal cysts by ultrasonography is mandatory to avoid confusion with primary renal hypoplasia associated with fibrosis and cysts, nephronophthisis being the most pertinent example. The presence of VUR and/or ureteropelvic junction obstruction (UPJO) does not automatically exclude the diagnosis of hypoplasia, as both conditions are frequently associated with primary renal-size defects. It is clear that this problem is difficult to resolve if the ureteral defect presents ipsilateral to renal hypoplasia. For example, severe antenatal hydronephrosis due to UPJO can determine the involution of the renal parenchyma and lead to an erroneous diagnosis of primary renal agenesis after birth. In bilateral cases, syndromic traits as well as inherited disorders such as medullary cystic kidney disease/nephronophthisis have to be excluded. Unequivocal exclusion of renal dysplasia is usually not feasible except in rare cases for which histology is available. It is possible that in the near future, molecular genetic advances could modify our present understanding and allow for a more direct separation of the two pathological entities based on laboratory tests.These challenges in clinical diagnosis of renal hypoplasia complicate studies attempting to discover new genes underlying this anomaly. For research purposes, we utilize a tentative classification scheme for categorizing our subjects for genetic studies: (1) isolated bilateral hypoplasia/dysplasia, (2) isolated unilateral hypoplasia/dysplasia, and (3) hypoplasia/dysplasia associated with lower tract abnormalities such as VUR or UPJO. Once the genetic basis of different subsets of urinary tract malformations is identified, the classification will likely be retrospectively changed and improved.The development of mammalian kidney derives from reciprocally inductive events between two tissue compartments of the embryonic metanephros: the ureteric bud (UB), an outgrowth of the nephric duct, and the MM. The ureteric bud invades the metanephric blastema at embryonic day 10.5–11 in the mouse and 35–37 in humans. The MM induces the ureteric bud to grow and branch while the ureteric bud induces the MM to transdifferentiate and form the nephrons’ epithelia (see recent reviews in kidney development in human and mice ).RET and Wingless-related 11 (WNT11) that are well recognized UB-specific molecules, whereas glial cell-line-derived neurotrophic factor (GDNF), Wilms tumor 1 (WT1), and Eyes absent 1 (EYA1) represent important examples of MM-specific factors. The paired-box gene 2 (PAX2) appears to be expressed in both structures during kidney development [GDNF signaling through the RET receptor is one of the best studied pathways, representing a critical step in the normal growth and branching of the UB during kidney development [Gdnf/Ret signaling has been shown to be the downstream mechanism underlying impaired nephrogenesis in many other mutant models . Numerous factors other than the Gdnf/Ret pathway also participate in kidney and urologic development , as evidenced by the long list of mutant mice with malformations in the kidney and urologic tract . SeveraIt is well known that nonsyndromic renal malformations may occur as hereditary traits and can present with familial aggregation. Evidence in favor of a genetic determination of the disease is raised by an increased recurrence risk among first-degree relatives and by several reports of familial occurrence of multiple malformations, including renal agenesis/hypoplasia and dysplasia. The relative recurrence risk of bilateral and unilateral agenesis has been estimated at 4–9% , 16, 17.Strategies to find genes causing renal agenesis/hypoplasia and dysplasia vary significantly depending on the characteristics of the study population available. Different data sets of patients have potential advantages and possible pitfalls.https://doi.org/www.hgmd.cf.ac.uk/ac/index.php) and most of the genes that are known contribute to susceptibility to common diseases [PAX2 and TCF2 mutations [RET enhancer has recently been shown to be a strong risk allele for Hirschsprung disease, explaining the paucity of coding mutations found in families showing linkage to the RET locus [So far, candidate gene studies have been the only alternative to linkage analysis to find genes underlying both Mendelian and complex traits. Such studies have identified many genes causing rare genetic diseases  .The genome-wide linkage analysis/positional cloning approach is a time-tested method used to identify disease-causing mutations, and it has been extremely successful in the past few decades for mapping genes that underlie monogenic Mendelian diseases , 33. ThiFor renal agenesis/hypoplasia and dysplasia, large pedigrees amenable for linkage analysis are very difficult to ascertain because these traits have incomplete penetrance . Moreover, many malformations, such as unilateral agenesis can be clinically silent and will not be detected without systematic screening of family members. As for candidate gene studies, locus heterogeneity is another potentially complicating factor that may dilute the power of linkage studies. Our previous data demonstrated that in the setting of reduced penetrance, variable expressivity, and very high genetic heterogeneity, approaches based on a limited number of uniquely large pedigrees or a very large number of medium-sized kindreds, are more likely to be successful to map a disease gene . As a reGenetic isolates represent a population structure that can greatly facilitate gene identification efforts. The genetic isolates are populations that are originated from a limited group of founders with little subsequent immigration into the population. Without an inflow of genes, a long period of time would be required for spontaneous mutations to rebuild genetic diversity. Therefore, genetic isolates are likely to harbor few disease-contributing alleles that have been inherited identical by descent from common ancestors –38. Theshttps://doi.org/www.ncbi.nlm.nih.gov/projects/SNP/) now contains about 5 million SNPs, including most of the SNPs with a minor allele frequency higher than 1% estimated to exist in the human genome [TCF7L2 have been found to confer risk to type 2 diabetes in different populations [The genome-wide association study is an approach aimed at exhaustively covering the genome to look for causative variants. Similar to genome-wide linkage studies, no assumptions are made about either the location of the causative variant or the biological role of the disease gene. Therefore, this approach represents an unbiased method to find disease-causing genes, with also a very high probability of discovering new genes, thus unraveling new pathophysiological pathways. Genome-wide association studies were not feasible until now because of the lack of information about the variability in the human genome and lack of low-cost, high-throughput genotyping technology. This situation has changed in the past 2 years: dbSNP  array allowed the discovery of the gene ry tract . CarefulRenal agenesis/hypoplasia and dysplasia still represents a challenge for both the clinicians who attempt a precise diagnosis and for the geneticists who try to unravel the genetic basis. Genetic and clinical approaches are now converging toward a common goal, which is the discovery of genetic markers, to make the diagnosis of this trait easier. The final objective is to improve classification, to make a reliable prognosis, and to attempt prevention. Based on advances from the last few years, the goal appears to be more feasible with large multicentric collaborative groups that share the same objectives and resources.


Several studies have suggested an association between periodontal disease and prematurity but this finding has not been consistently observed.Case control study. Cases (n = 50) were women who had delivered after spontaneous preterm labor at <35 weeks gestation. Two groups of controls (n = 101) were recruited: women who were undelivered but at a preterm gestation and women who delivered at term. A standard, clinical, periodontal examination was performed and gingival crevicular fluid was obtained from standardized locations and tested for neutrophil elastase along with the bacterial enzymes gingipain and dipeptidylpeptidase. Data were analyzed with Fisher's exact tests, ANOVA and multivariate logistic regression.ul vs. 159.6 uU/ul p = .007 and 2.70 uU/ul vs. 1.56 uU/ul p = .001. On multivariate analysis, the mean log concentration of elastase, but not of gingipain, remained a significant predictor of preterm labor p = .0.015.There was no difference in the proportion of sites with significant attachment loss (≥3 mm): Cases-3.2%, Controls-2.2% p = 0.21. The gingival crevicular fluid concentrations of elastase and gingipain were elevated in cases vs. controls 238.8 uU/We found no evidence that clinical periodontal disease is associated with spontaneous preterm birth. Elevated gingival crevicular fluid levels of elastase were associated with preterm birth but further research is needed before this can be assumed to be a causal relationship. Preterm birth remains the most important cause of perinatal mortality and morbidity. Despite considerable research, the pathogenesis of preterm delivery is not well understood and no effective preventative therapy is available. Several, recent studies have suggested a relationship between preterm delivery and periodontal disease. Offenbacher et. al. reported a strong association, OR = 7.9, between periodontal disease and adverse pregnancy outcome defined as either premature delivery or low birth weight in women in North Carolina . SimilarOne concern with these studies is that confounding, especially by socioeconomic status, has not been consistently controlled for. Socioeconomic status has been associated consistently with spontaneous preterm birth and may also be associated with poor dental care, particularly in jurisdictions where it is not publicly funded. As well, no studies of which we are aware, related to spontaneous premature birth, have evaluated biochemical measures of active periodontal disease in addition to standard clinical examination. Since periodontal disease is characterized by a relapsing/remitting pattern, identifying active disease may be an important factor in establishing associations with other disease states such as preterm labor. The population from which the subjects were recruited is also high risk for premature delivery. The city where this study was performed has one of the highest rates of preterm birth in Canada with 10% of births in 2004 occurring before 37 weeks . The population is predominately Caucasian (81%) with Chinese (6%), South Asian (4%), Aboriginal (2%) and Black 1.5%) comprising the other main ethnic groups [% comprisPeriodontitis is an inflammatory process initiated by bacterial plaque involving the supporting structures of the tooth which include the gingiva, the junctional epithelium, root cementum, periodontal ligament, and alveolar bone. These sThis case control study was designed to assess the possible relationship between periodontal disease and spontaneous preterm delivery using both clinical examination and the assessment of neutrophil elastase, bacterial gingipain and dipeptidylpeptidase in gingival crevicular fluid.Cases were women with singleton pregnancies who delivered at or before 35 weeks gestation, either after spontaneous labor or induction of labor for preterm premature rupture of membranes. The subjects all had access to universally funded prenatal care but not to publicly funded dental care. Two groups of controls were recruited: a group of postpartum women who delivered at term and the other, undelivered women who were assessed between 22 and 35 weeks gestation. Women were recruited in one hospital following delivery and in the hospital antenatal clinics.uL of 50 mM 2-(N-morpholino)-ethane-sulphonic acid (MES) buffer, pH 5.5, with 0.1 mM dithiothreitol (DTT), 0.15 M NaCl and 0.1% v/v Triton X-100. After 1 hour, with occasional agitation at 4°C, the strips were removed and the eluates were frozen with dry ice. The samples were maintained at -20–70°C and transported for analysis in the laboratory of two of the co-investigators  Enzyme activities were determined using selective peptide substrates for neutrophil elastase , bacterial gingipain  and dipeptidylpeptidase (Ala-Pro-AFC). Assay procedures and conditions were as described previously for each enzyme[Consenting subjects had a full periodontal examination in a standard, well- equipped, dental office maintained by one of the co-investigators, an experienced periodontist (AF). The dental examination was performed between 2 days and 28 days following delivery for the post partum subjects. Clinical examination was carried out by one highly experienced periodontal hygienist (S.N.). The intra-person variability in clinical examination by the hygienist was assessed by one of the investigators (AF) in a pilot period, using five volunteers, and was found to be acceptably low (mean .34 mm SD .5 mm). Clinical examination included assessment of oral hygiene with a standardized index   and probch enzyme ConcentrStandardized questionnaires were used to obtain data relating to medical-dental history and socioeconomic status.Two control groups were used in this study as a previous investigation of periodontal disease in pregnancy had documented decreases in probing depth at term compared to preterm gestations . TherefouU/ul, bacterial dipeptidylpeptidase >30 uU/ul and gingipain >30 uU/ul. Analysis was performed with Stata version 8. A sample size of 50 cases and at least 50 controls was estimated to have at least 80% power to detect an association between clinical periodontitis and preterm birth of the magnitude of an odds ratio equal to 4. The sample size calculation was also based on an estimate of a 13% prevalence of periodontal disease [Univariate analysis was planned to evaluate mean attachment loss and the frequency of significant attachment loss, (3 mm), between the cases and controls. Final analysis with logistic regression was planned to include possible confounding factors such as smoking, income, and education. Univariate analysis of the log mean enzyme concentrations was also planned as well as a comparison of the number of subjects who had concentrations over critical enzyme levels as this previously had been shown to predict attachment loss ,9: neutrThe study was approved by our institutional ethics review board.During the study, we recruited 151 women, 50 cases, 51 undelivered controls and 50 postpartum term controls. One of the undelivered controls delivered preterm, two weeks after her dental examination, so she was reassigned as a case. The remainder of the undelivered controls all subsequently delivered at term.The 50 cases delivered after spontaneous preterm labor between 22 and 35 weeks gestation (mean = 30.8 +/-3.7 weeks). The mean gestational age of the undelivered controls on examination was 29.2 +/- 4.2 weeks. The post partum controls delivered at a mean gestational age of 39.4 +/-1.1 weeks. Additional characteristics of the subjects are described in Table Initial analysis of the attachment loss of the two control groups revealed no significant differences so, as planned, for the final analysis, they were combined. The percentage of sites with attachment loss ≥ 3 mm was 2.2% in the combined control group compared to 3.2% in the preterm cases p = .21. It was originally intended to dichotomize the subjects with an extent severity score of , which was used by Offenbacher . An exteuU/ul , compared to 30 undelivered controls  and 32 postpartum controls . Mean log concentrations of gingipain were higher in the cases compared to undelivered and postpartum controls, p = .005 (Table uU/ul) were observed in 6 preterm cases, three subjects with one high enzyme site, one subject with two and two subjects with three high enzyme sites. Three undelivered controls and one postpartum control subject had one site each with high gingipain levels. There was no significant difference in the mean log concentration of bacterial dipeptidylpeptidase or in the frequency of sites with critical levels between the cases and controls  p = 0.015, but the association with gingipain concentration did not: OR= 3.06  p = .15. The multivariate analysis was also performed replacing enzyme concentration with a variable for the number of sites with high enzyme levels. The number of high elastase sites was significantly associated with spontaneous preterm birth OR = 1.25 , p = 0.021. The number of high gingipain sites was also positively associated with premature birth but, again, did not reach statistical significance OR = 1.43 , p = 0.61. Although the lack of dental cleaning within 6 months of delivery had an association with premature birth in univariate analysis, the association was no longer statistically significant in the final model OR = 1.15 , p = 0.43. Further analysis was then performed to determine if high neutrophil elastase was associated with dental history. It appeared that having a dental cleaning within 6 months and not smoking during pregnancy were highly associated with lower gingival crevicular fluid elastase concentrations . Additional regression analysis was also performed to confirm that the time from delivery to dental examination was not associated with neutrophil elastase levels p = .28.Our study did not find an association between clinical periodontitis, measured by attachment loss, and spontaneous preterm birth. This is in contrast to several of the studies to date ,2,15,16 Another explanation for a negative finding in any observational study is measurement error. We consider it quite unlikely that this could be responsible for our findings as we used only one, highly trained, blinded examiner to assess all the patients. This consistency in examination should reduce variability in measurement and any tendency to bias the results to a null finding. Finally, limited power could be an explanation for our findings. Although our study was relatively small, the degree of attachment loss was almost identical amongst the three groups. In order to detect a statistically significant difference, of the magnitude we observed, we would have required over 8000 subjects. Our study was also more than adequately powered to detect the strong associations that have been reported in the literature to date, especially if our study had observed a similar frequency of severe periodontal disease as those studies. The fact that we did not find the same frequency of disease does not invalidate our findings and suggests that an association between clinically measured periodontal disease and prematurity may not be evident in all populations. However, we do recognize that, given the relatively small size of our study, inadequate power is always a possible explanation for a null finding.However, we do report an association between a marker of active periodontitis, gingival crevicular fluid neutrophil elastase, and premature delivery. It is difficult to fully evaluate the significance of this finding, especially as we did not find an association between premature delivery and standard clinical measurements of periodontitis. In our study, the gingival crevicular fluid enzymes were measured after delivery. It may be that following a preterm delivery, women are under increased stress and may neglect their dental hygiene. As neutrophil elastase may rise in gingival crevicular fluid with the development of gingivitis, this may be all that is reflected in our data. However, analysis of the Oral Hygiene Index scores between the case and control groups suggested only a slight, non-significant increase in only the debris scores, but not in the calculus scores, in the preterm group. The possibility of selection bias should also be considered in the evaluation of the results of any case control study. However, none of our patients had symptomatic periodontal disease. Therefore, it seems difficult to conceptualize how patients or study nurses could have perceived factors associated with elevated gingival crevicular fluid enzyme levels and have let this influence the probability of enrolment. One explanation for the inconsistency between the clinical and enzyme data is that high elastase levels reflect active periodontal inflammation and this, rather than past attachment loss, is the more important risk factor. If so, it may be that, in our minimally diseased population, the subjects have not had time to developed clinically apparent disease. Based on our data, such a conclusion is reasonable and would be consistent with a recent publication documenting an increased risk of preterm birth with progression of attachment loss during pregnancy . FurtherIt is also possible that previous studies and our own enzyme findings have simply detected a yet to be defined generalized susceptibility to infection in women who deliver preterm. This could place women at increased risk for a variety of infections including periodontal disease and ascending chorioamnionitis leading to premature labor. On the other hand, one possible basis of shared risk, cytokine gene polymorphisms, does not appear to be a common factor in preterm birth and periodontitis .In conclusion, in contrast to other authors, we did not find an association between clinical periodontal disease, measured by attachment loss, and spontaneous premature delivery. We did find an association between gingival crevicular fluid enzyme levels and premature delivery, possibly indicating an association between active periodontal disease and spontaneous premature delivery. However, given the lack of agreement between the analysis of the clinical and enzyme data, we feel it would be premature to conclude that a causal relationship exists. Ultimately, because of the difficulty in proving a temporal relationship between exposure and disease, a case control study can rarely, in and of itself, prove causality. Therefore, at this time a recommendation for screening and treatment of periodontal disease in pregnant women, with the aim of preventing premature birth, cannot be made until further research is completed. Of course, it is still recommended that women who are pregnant or planning to become pregnant continue to maintain optimum periodontal health with professional cleanings and meticulous oral hygiene to prevent periodontal disease. Based on our results, future investigators in this area should also consider measuring markers of active periodontal disease and not rely solely on clinical examination.The author(s) declare that they have no competing interests.All authors contributed to the design of the study. AF, RS and SW conceived the study. SW supervised the recruitment. SN carried out the periodontal examinations under the supervision of AF. SC and BM carried out the GCF enzyme analysis. RB performed the statistical analysis. All authors contributed to and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The use of cellular and cordless telephones has increased dramatically during the last decade. There is concern of health problems such as malignant diseases due to microwave exposure during the use of these devices. The brain is the main target organ.Since the second part of the 1990's we have performed six case-control studies on this topic encompassing use of both cellular and cordless phones as well as other exposures. Three of the studies concerned brain tumours, one salivary gland tumours, one non-Hodgkin lymphoma (NHL) and one testicular cancer. Exposure was assessed by self-administered questionnaires.Regarding acoustic neuroma analogue cellular phones yielded odds ratio (OR) = 2.9, 95 % confidence interval (CI) = 2.0–4.3, digital cellular phones OR = 1.5, 95 % CI = 1.1–2.1 and cordless phones OR = 1.5, 95 % CI = 1.04–2.0. The corresponding results were for astrocytoma grade III-IV OR = 1.7, 95 % CI = 1.3–2.3; OR = 1.5, 95 % CI = 1.2–1.9 and OR = 1.5, 95 % CI = 1.1–1.9, respectively. The ORs increased with latency period with highest estimates using > 10 years time period from first use of these phone types. Lower ORs were calculated for astrocytoma grade I-II. No association was found with salivary gland tumours, NHL or testicular cancer although an association with NHL of T-cell type could not be ruled out.We found for all studied phone types an increased risk for brain tumours, mainly acoustic neuroma and malignant brain tumours. OR increased with latency period, especially for astrocytoma grade III-IV. No consistent pattern of an increased risk was found for salivary gland tumours, NHL, or testicular cancer. During the most recent decades there has been a rapid development of the use of wireless telephone communication. The Nordic countries in Europe were among the first in the world to introduce this new technology.The analogue  phones operating at 450 MegaHertz (MHz) were introduced in Sweden in 1981. In the beginning they were usually used in a car with fixed external antenna. Portable NMT 450 phones were introduced in 1984. Analogue phones using 900 MHz (NMT 900) were used in Sweden between 1986 and 2000. The digital system  started in 1991 and has during recent years dramatically increased to be the most common phone type. This system uses dual band, 900 and 1 800 MHz, for communication. From 2003 the third generation of mobile phones, 3G or UMTS  has started in Sweden operating at 1 900 MHz.Desktop cordless phones also use wireless technology. First the analogue system in the 800–900 MHz RF was used when these phones were available in Sweden in 1988. Digital cordless telephones (DECT) that operate at 1900 MHz are used since 1991.Use of mobile and desktop cellular telephones results in exposure to microwaves. Exposure is characterized through the specific absorption rate (SAR) expressed as watt/kg. The anatomical area with the highest exposure is the ipsilateral (same) side of the brain that is used during the call. If a hands-free device is used and the cellular telephone is placed at another part of the body that anatomical area receives the highest radio frequency (RF) exposure. The cellular telephone communicates with a base station usually located at some distance, the antenna of which typically is on the top of a building or on a mast. Several workplaces use only cellular or cordless phones instead of the landline phones and this leads to both active and passive exposure to microwaves during the working day for the employees. Very few workplaces offer hands free devices to the employees although the Nordic radiation protection authorities as well as the Swedish work environmental board recommend this.The introduction of wireless communication has been technically driven without proper laboratory testing or epidemiological studies of potential health effects. Among the first to express concern of adverse health effects due to exposure to microwaves from cellular phones was the layman . At thatSince the second part of the 1990's we have performed six case-control studies on this topic encompassing use of both cellular and cordless phones as well as other exposures. This is an overview of the findings in these studies. Three of our studies concerned brain tumours. The first one was rather small ,3. This During the same time we studied risk factors for non-Hodgkin lymphoma (NHL), mainly to elucidate pesticide exposure as discussed elsewhere . In thatFinally we have also studied testicular cancer, the main topic being chemical exposures, e.g., polyvinyl chloride . The resIn the following a short description of the studies is given, further details are displayed in the various publications. In principle the same epidemiological methods were used in all studies.All studies were performed in Sweden covering various health service regions and at somewhat different time periods for recruitment of cases and controls, see Table All investigations were approved by the responsible ethical committees and were performed according to the ethical standards laid down by the Helsinki Declaration. All included persons had the possibility to refuse participation. Exposures were assessed by mailed questionnaires and the answers were supplemented over the phone by a trained interviewer using a structured protocol. The interviews as well as coding of the answers for statistical analyses were made blinded as to case or control status. Details have been further explored in the various publications. It should be noted that use of cordless phones was not assessed in our first brain tumour study ,3.Odds ratios (OR) and 95 % confidence intervals (CI)  were calculated using conditional logistic regression analysis in the first study on cellular telephones and brain tumour risk ,3. In thIn the study on NHL adjustment was made for age, sex and year of diagnosis (cases) or enrolment (controls). The results in the testicular cancer study were adjusted for age and cryptorchidism.Latency or tumour induction period was in this presentation analysed using three time periods, > 1 year, > 5 years and > 10 years since first use of a cellular or cordless telephone until diagnosis. In the dose-response calculations median number of cumulative lifetime use in hours among controls was used as cut-off. Regarding brain tumours calculation of trend was made dividing cumulative use among the controls in tertiles.The response rates in the different studies were high, see Table In our first study no increased risk was found overall, see Table The following two case-control studies on brain tumours were larger and encompassed answers from 1 254 88 %) of cases with benign brain tumour, 905 (90 %) with malignant brain tumour and 2 162 (89 %) controls. Here results are given from the pooled analysis of these two case control studies 8 % of ca.Regarding meningioma the risk increased with latency period. With latency > 10 years analogue phones yielded OR 1.6, 95 % CI 1.04–2.6, digital phones OR 1.8, 95 % CI 0.7–4.6 and cordless phones OR 1.8, 95 % CI 1.01–3.2. However, in the multivariate analysis adjusted for the different phone types lower ORs were found and no was statistically significant . For seminoma significantly increased OR was calculated in lowest exposure category with > 1 year latency period for all studied phone types. However, there was no dose-response effect and no significant trend for increasing OR with increasing latency period. As one would expect cryptorchidism was associated with increased risk for both seminoma and non-seminoma but did not interact with use of cellular or cordless phones. The localization of the mobile phone during stand-by time was also analyzed. However, no association was found with testicular cancer. Keeping the phone in a pocket close to the testis did not increase the risk and there was no association with laterality of the phone and cancer.In studies of tumour risk and mobile phone use exposure assessment becomes an even greater problem than for the acute effects since for this type of disease it is the exposure 5–10 years or more ago that is of interest. Most users of mobile phones have not been using just one single telephone. It is even more likely that if they have been using a mobile phone for more than a few years, they will also have changed their phone a few times. Many users will also have used different phone systems such as analogue and digital, and probably many of them have also been using a cordless phone at home or at work. The problem we are facing is then how to integrate the various SAR distributions from the different devices and add up the different times on these phones to one exposure measure? At the moment it is not clear how to combine the use of different phones with different power output, different systems, different frequencies, and different anatomical SAR distribution, into one exposure and dose measure. The difficulties lay in the fact that we do not know the interacting mechanism(s) between the electromagnetic fields emitted from the phone and the biological organism.We used a weighting method as described above to combine exposure measurement from different phone types; NMT = 1, GSM = 0,1 and cordless phones = 0.01. This method was applied for data in our second brain tumour study ,5 and haIn future epidemiological studies on brain tumours an important consideration ought to be which time scale to use, and this must be based on hypotheses about induction and progression of the endpoint variables being studied. One needs to set up a clear hypothesis about how the absorption to RF from mobile phones could influence the endpoint variable in terms of anatomical localization of the absorption, the duration of the exposure and the induction and progression of the endpoint variable before choosing an appropriate dosimetric quantity.One of the questions we need to address is for instance how time comes into the connection between exposure and dose, and here we need to distinguish between different aspects of time: very short times – order of minutes, daily averages, and total time in the actual occupation – number of years with exposure. Another question that is urgent to address is the potential for greater biological effects from RF fields in young age groups. We have found some indication for that with higher risk for brain tumours in persons with first use of cellular or cordless phones before the age of 20 years compared with older ages ,9,22.We have here presented results from our studies on this topic. The intention was not to cover the whole area, such presentations can be found in other publications -25. In oThe author(s) declare that they have no competing interests.LH was the principal investigator responsible for the design, conduct and interpretation of the studies.KHM participated in all aspects of the studies, especially with his technical knowledge.MC participated as a statistician in all parts of the studies.FS participated in the compilation and interpretation of the data for this publication.


However, previous studies of the ACE-I/D variant and preeclampsia have been individually underpowered to detect plausible genotypic risks.Inappropriate activation of the renin–angiotensin system may play a part in the development of preeclampsia. An insertion/deletion polymorphism within the angiotensin-I converting enzyme gene ACE-I/D polymorphism. Crude and adjusted odds ratio (OR) and 95% confidence interval (CI) using logistic regression models were obtained to evaluate the strength of the association between ACE-I/D variant and preeclampsia risk. A meta-analysis was then undertaken of all published studies to February 2006 evaluating the ACE-I/D variant in preeclampsia. An additive model  revealed a null association between the ACE-I/D variant and preeclampsia risk  in the new case-control study. Similar results were obtained after adjusting for confounders  and using other genetic models of inheritance. A meta-analysis  showed a per-allele OR of 1.26 . An analysis stratified by study size showed an attenuated OR toward the null as study size increased.A prospective case-control study was conducted in 1,711 unrelated young pregnant women  recruited from five Colombian cities. Maternal blood was obtained to genotype for the ACE D-allele is due to small-study bias, similar to that observed in cardiovascular disease. Reliable assessment of the origins of preeclampsia using a genetic approach may require the establishment of a collaborating consortium to generate a dataset of adequate size.It is highly likely that the observed small nominal increase in risk of preeclampsia associated with the  ACE D-allele is likely to be due to small-study bias, a similar result to that observed in cardiovascular disease.The observed small increase in risk of preeclampsia associated with the Preeclampsia is a common condition affecting pregnant women worldwide; it is defined as the presence of increased blood pressure, together with protein in the urine. Although in many women preeclampsia may never result in symptoms, other women may experience headaches, problems with their vision, swollen ankles and feet, and other problems. Sometimes, preeclampsia progresses to eclampsia, in which potentially life-threatening seizures result. The causes of preeclampsia are not well understood, but several factors are known to contribute to the risk. These factors include diabetes, high blood pressure prior to pregnancy, obesity, and first pregnancy. There is also the possibility that preeclampsia has, at least in part, a genetic basis; the condition is more likely among women whose relatives have also had it. However, no definite genetic cause has yet been confirmed.ACE, which codes for the angiotensin-1 converting enzyme, has been linked with preeclampsia in a number of different studies. The protein encoded by ACE is involved in controlling blood pressure and the balance of fluid and salts in the blood. However, many of the studies supposedly linking ACE and preeclampsia were done on very few participants. Small studies are more likely to generate “false positive” findings. Therefore, a group of investigators from Colombia and the UK wanted to find out whether they could reproduce the supposed link between the ACE gene variant and preeclampsia in a large study, and also to see whether the previous studies could have been “false positives.”A common variant in one particular gene, ACE gene. The investigators then did a statistical comparison to see whether there was any association between preeclampsia and possession of a particular variant of the ACE gene. The results showed that there was no such association. Then, the investigators did a literature search to find all previous studies that had examined a possible link between variants of the ACE gene and preeclampsia. They found 22 studies reporting data obtained from 6,424 women (these figures include the results from the investigators' own case-control study described here). The data from all of these studies were then put together into a combined analysis. This combined analysis did suggest a small increase in the risk of preeclampsia in women with one particular variant in the ACE gene. However, this result was more likely in studies with small numbers of participants. Furthermore, the earliest studies done were most likely to show an effect, with the supposed link disappearing as more and more data were collected.These investigators carried out a case-control study. This means that women with preeclampsia (“cases”) were recruited, and compared with women similar in all other respects but who did not have preeclampsia (“controls”). In total 1,711 pregnant women from five Colombian cities were studied, of whom 665 had preeclampsia and 1,046 did not. Blood was taken from each participant and used for DNA sequencing of the ACE gene variants and preeclampsia is a spurious one. The investigators propose that in future, collaborative research networks will be needed to carry out rigorous research on the genetics of preeclampsia. Such initiatives will help to overcome the problem of bias that can arise from small studies.The findings presented here suggest that “small study bias” may explain the discrepancy between the results of the case-control study and the combined analysis. That is, studies involving few participants are less reliable and more likely to produce false-positive results. Therefore, it is possible that the proposed link between http://dx.doi.org/10.1371/journal.pmed.0030520.Please access these Web sites via the online version of this summary at  NHS Direct  about preeclampsiaInformation for patients fromMedLine Plus, supplied by the US National Library of MedicineMedical encyclopedia entry on preeclampsia from maternal health in the AmericasInformation from the World Health Organization and Pan American Health Organization on  Preeclampsia is a maternal disease of pregnancy associated with increased blood pressure and proteinuria after 20 weeks of gestation. It is a major cause of maternal and neonatal mortality and morbidity worldwide and has particularly high incidence in Latin American and Caribbean countries, in which hypertensive disorders during pregnancy account for 25.7% of maternal deaths ,2. PreecWith the exception of smoking, established risk factors for cardiovascular disease, including high blood pressure, diabetes, and obesity, are also risk factors for preeclampsia . In addi(ACE-I/D) has been reliably associated with substantial differences in the plasma and tissue angiotensin-converting enzyme (ACE) activity in a codominant (additive) fashion not only in persons of European descent, but also in other populations such as Hispanics [D allele have higher ACE activity, which has been proposed as an intermediate phenotype of potential relevance for the development of high blood pressure and subclinical atheroma  [ACE-I/D polymorphism on ACE activity, associations of the ACE-I/D polymorphism and coronary heart disease, coronary artery restenosis, stroke, and renal disease have been inconsistent [Inappropriate activation of the renin–angiotensin system may play a part in the development of many cardiovascular disorders, including preeclampsia ,8. A comispanics –11. Indi artery) ,12. Despnsistent –16. Morensistent –16.D allele of the ACE-I/D polymorphism have higher ACE activity and higher measures of uterine artery resistance, which is a marker for development of intrauterine growth retardation and preeclampsia [ACE-I/D polymorphism may be a good candidate in the search for a cause of preeclampsia. However, to date, studies evaluating the role of ACE-I/D polymorphism in preeclampsia have been individually underpowered to detect plausible genetic effect sizes, being much smaller than more recent studies in cardiovascular disease. We hypothesized that the published literature on the ACE-I/D polymorphism in preeclampsia might be similarly affected by small-study bias. To test this hypothesis, we conducted a new large genetic association study on the ACE-I/D polymorphism and preeclampsia in a geographical region with a high incidence of preeclampsia. We then set this study within the context of a systematic review and a meta-analysis of all studies conducted to date.Several studies, also usually small in size, have reported that women carrying the clampsia ,17. ThesA prospective case-control study was conducted in 1,711 unrelated young pregnant women  recruited from five Colombian cities at the time of delivery between January 2000 and December 2005. A verbal interview with a structured questionnaire was conducted by trained personnel at the time of the delivery to ascertain maternal age, gestational age, parity, smoking status during pregnancy, family history of preeclampsia, ethnic background, and socioeconomic position. On two separate occasions, the mean of two readings of blood pressure was obtained  at the time of delivery. Blood pressure was measured in the right arm after a five-minute period of rest in a seated position using mercury sphygmomanometers or electronic devices calibrated against a mercury standard.A case was defined as a primigravid woman younger than 26 years old with blood pressure 140/90 mm Hg or above, and proteinuria 0.3 grams or above in 24 hours, or a reading 2+ or above on a dipstick in a random urine determination with no evidence of urinary tract infection after 20 weeks of gestation . At leasACE-I/D polymorphism in intron 16 was detected according to the method described by Rigat, et al. [(D) allele in contrast to the larger insertion (I) allele in a competitive amplification reaction when both alleles are present, as occurs in individuals with the ID genotype. This leads to mistyping of ID individuals as DD in approximately 4%–5% of the samples. To avoid any mistyping of ID as DD, a second PCR amplification using insertion-specific primers was conducted for all participants who were homozygous for the D allele [n = 156) was subjected to a second PCR and genotyping to minimize any possible misclassification. The Cohen's kappa value among the samples regenotyped was equal to 0.95 , and the error rate was 2.56% . Genotyping was conducted blinded to the clinical status of the participants.Blood was drawn from the antecubital vein into EDTA and samples stored at −50 °C for DNA extraction, using the QIAamp DNA blood mini-kit . The , et al. . PCR is D allele . For a d2, or Mann-Whitney tests were used as appropriate. A test for departure from Hardy-Weinberg equilibrium was performed by χ2 analysis. The principal a priori hypothesis was that the association between the ACE-I/D polymorphism and preeclampsia follows an additive model according to the number of D alleles. The additive “per-allele” model  was based on the effect of the ACE-I/D variant on its intermediate phenotype (ACE activity), established in large studies and overviews as being a reliable association [ID versus II and DD versus II) were also evaluated for completeness as secondary outcomes.To evaluate the presence of differences between groups, unpaired Student's t-, χociation . HoweverMultivariate analysis using logistic regression methods was also conducted to control for potential confounders .To explore the prior hypothesis that the genotypic odds ratio (OR) is greater in women with an enriched phenotype, an analysis using an additive model was repeated for women stratified according to the presence of family history of preeclampsia (positive versus negative family history in mother or sisters), and disease severity with severe preeclampsia being defined as blood pressure above 160/110 mm Hg or proteinuria of 5 grams or more in 24 hours, eclampsia, or the HELLP syndrome. For these analyses 99% CIs were used to make some allowance for their exploratory nature. All statistical analyses were conducted using Stata, Version 9 .ACE-I/D polymorphism and preeclampsia in humans in all languages. The search strategy contained both medical subject heading terms and text words as follows: “angiotensin-converting enzyme” or “ACE” or “peptidyl-dipeptidase A,” in combination with “pre-eclampsia” or “preeclampsia” or “pregnant hypertensive disorders” or “pregnancy hypertension,” and combined with “genetic” or “polymorphism(s)” or “mutation” or “genotype” or “gene(s).” No limits were used in the search strategy. We searched for any additional studies in the references of all identified publications, including previous relevant meta-analyses, and used the MEDLINE option “related articles” for all the relevant papers.Electronic databases  were searched up to February 2006 for all genetic association studies evaluating the ACE-I/D polymorphism and preeclampsia. Studies published as full-length articles or letters in peer-reviewed journals in any language were included, as well as abstracts taken from reference lists of identified publications. Authors were contacted (on at least three occasions) to obtain information on the genotype frequency by case-control status and by disease severity (severe and nonsevere preeclampsia), the use of blinding of genotyping staff to clinical status, the definition of outcomes, and, in a few cases, to clarify possible overlapping of study results. A positive reply was obtained in 15 out of 21 study authors contacted.For inclusion, studies had to involve unrelated women and examine the association between the χ2 goodness of fit for Hardy-Weinberg equilibrium and its p-value, use of blinding, mean age of participants, frequency of nulliparous women, and main exclusion criteria. In the few instances in which genotype frequencies provided by the investigators in tabular data differed slightly from published figures, the tabular data were used. Women with gestational hypertension were excluded from the present meta-analysis in order to improve the homogeneity of phenotype between studies.The following information was extracted  from each study and disagreements resolved by consensus: year of publication, total cases, total controls, number of individuals by each genotype, study design, source of controls, matching variable, thresholds used to define preeclampsia, country of origin, ethnicity, D allele was considered the one at risk. For the additive or per-allele model, the OR was compared between cases and controls by assigning scores for different genotype groups and calculating ORs by logistic regression. We calculated the random effect summary OR and CIs for each polymorphism. To make some allowance for multiple comparisons 99% CIs were used for individual studies, and 95% CIs were reserved for the combined estimates. The inverse variance-weighted method was used to calculate the summary OR [Q test and I2 was used as a measure to describe the percentage of variability in point estimates that was due to heterogeneity rather than sampling error [Data were analysed using Stata 9. The genetic model to be considered as the priori hypothesis was an additive model, following the considerations previously described for the case-control study. Secondary analyses involved ORs for other genetic models of inheritance such as recessive, dominant, and pairwise comparisons of the different genotypes. For all the models used, the mmary OR . Heterogng error . Sourcesng error ,21. To eng error .Clinical and demographic data of the cases and controls are shown in ACE-I/D genotypes and allelic frequencies were not significantly different according to the case-control status  revealed a null association between the ACE-I/D variant and preeclampsia risk . Adjusting for maternal age, ethnicity, recruitment centre, and place of birth, aimed to minimize the effect of possible population admixture, did not change the estimate of the effect . A similar result was obtained after further adjustment for additional potential confounders such as socioeconomic position, presence of urinary or vaginal infections during pregnancy, and smoking status during pregnancy . ORs for other genetic models of inheritance also yielded a null association , no substantial heterogeneity of the genetic effect size was observed for any of the subgroups  . Study characteristics such as blinding of genotyping staff, publication language, preeclampsia definition, and disease severity explained little of the heterogeneity  . Similarly, stratifying by ethnicity indicated that studies conducted in Asian populations tended to have a larger ORs . These findings might be explained by the fact that eight out of the nine studies conducted in Asian populations had fewer than 100 cases in each study. The funnel plot including all studies was asymmetric, and the Egger's test (p = 0.06) and the linear regression model (p = 0.003) suggested the presence of an excess of small studies with more positive results, predominately of studies published in non-English–language journals  . Howeverogeneity . A strat 0.0002) . AnalogoACE-I/D variant in preeclampsia. Although a pooled per-allele OR suggested evidence of an increase in the risk of preeclampsia of 1.26 , the robustness of this summary estimate is uncertain. First, our study found a null association of the ACE-I/D variant with preeclampsia. Moreover, there was no substantial evidence of a positive effect in any of the subgroups in the prespecified analyses. Second, the meta-analysis revealed diminishing summary risk estimate as study size increased (p = 0.06 and linear-regression model test p = 0.003). Furthermore, since only the published literature was included, it is possible that including unpublished studies (which more often provide evidence of negative or null effects) would have provided additional evidence of small-study bias. Taken together, these findings point to small-study bias as a potential explanation for the results observed in the meta-analysis. Discrepant findings from large and small studies are not new in the field of genetics of complex disorders [The current meta-analysis, which includes new data from the largest case-control study to date, represents the most comprehensive evaluation of the ncreased , regardlisorders . When prisorders . Furtherisorders .http://www.wtccc.org.uk) [Publication bias is increasingly being recognized as one of the main threats to the reliability of conclusions drawn from association studies with common disease outcomes. In the setting of cardiovascular and neurological diseases, several positive gene–disease associations, usually based on meta-analysis of small studies, have been subsequently refuted by large genetic studies ,53. As a.org.uk) . Genetic.org.uk) ,56 and a.org.uk) ,57. Howe.org.uk) . TherefoDespite these obstacles, the investment in adequate resources to study the genetics of preeclampsia is an important priority. Observational studies and the randomised trials of interventions that have followed have been unsuccessful thus far in identifying causal pathways in preeclampsia amenable to preventive therapies, a clear example of which are the recently failed clinical trials using either antioxidant vitamins or calcium supplements –61. A geInvestigating the aetiology of preeclampsia, one of the main causes of maternal and neonatal mortality and morbidity worldwide, should be a health research priority. A genetic approach may indeed be useful, but large collaborative studies will also be needed.Figure S1ORs for outcome using a per-allele model. Studies in bold are those published in non-English-language journals.(38 KB PPT)Click here for additional data file.Figure S2OR (random effect model) for outcome using a per-allele model.(36 KB PPT)Click here for additional data file.Protocol S1(68 KB DOC)Click here for additional data file.Alternative Language Abstract S1Translation by N. C. Serrano, Universidad Autónoma de Bucaramanga, Bucaramanga, Colombia.(25 KB DOC)Click here for additional data file.Alternative Language Abstract S2Translation by D. Wang, Medical Statistics Unit, London School of Hygiene and Tropical Medicine, London, United Kingdom.(33 KB DOC)Click here for additional data file.http://www.ncbi.nlm.nih.gov/SNP/) single-nucleotide polymorphism discussed in this paper is an insertion/deletion variant (rs1799752) referenced to the ACE gene with the GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) accession number J04144.The SNP database (


Chronic alveolar hypoxia results in sustained arterial constriction, and increase in pulmonary vascular resistance leading to pulmonary artery hypertension (PAHT). The aim of this study was to investigate the effect of propofol and etomidate on pulmonary artery (PA) reactivity in chronically hypoxic (CH) rats, a model of pulmonary arterial hypertension (PAHT), in normoxic animals, and human PA.t tests with Bonferroni correction as post-hoc tests.CH rats were maintained 14 days at 380 mmHg pressure in a hypobaric chamber. Human tissue was retrieved from histological lung pieces from patients undergoing resection for carcinoma. Cumulative concentrations of anaesthetics were tested on isolated vascular rings precontracted with phenylephrine (PHE) or 100 mM KCl. Statistical comparisons were done by ANOVA, followed, when needed, by Student max) induced by etomidate and propofol was 101.3 ± 0.8% and 94.0 ± 2.3%, respectively, in KCl-precontracted rings, and 63.3 ± 9.7% and 46.1 ± 9.1%, respectively, in PHE-precontracted rings (n = 7). In KCl-precontracted human PA, Rmax was 84.7 ± 8.6 % and 66.5 ± 11.8%, for etomidate and propofol, respectively, and 154.2 ± 22.4 % and 51.6 ± 15.1 %, respectively, in PHE-precontracted human PA (n = 7). In CH rat PA, the relaxant effect of both anaesthetics was increased in PHE-precontracted and, for etomidate only, in KCl-precontracted PA. In aorta, CH induced no change in the relaxant effect of anaesthetics.In normoxic rat PA, maximal relaxation (RPropofol and etomidate have relaxant properties in PA from human and normoxic rat. The relaxant effect is specifically accentuated in PA from CH rat, mainly via an effect on the pharmacomechanical coupling. Etomidate appears to be more efficient than propofol at identical concentration, but, taking into account clinical concentrations, etomidate is less potent than propofol, which effect was in the range of clinical doses. Although these findings provide experimental support for the preferential use of etomidate for haemodynamic stability in patients suffering from PAHT, the clinical relevance of the observations requires further investigation. Chronic hypoxia (CH) occurs in people living at high altitude and in children who suffer congenital heart disease with left to right shunt; it is also a main characteristic of chronic obstructive pulmonary disease, a major cause of death that affects a significant proportion of adults. Chronic alveolar hypoxia results in sustained arterial constriction, an increase in pulmonary vascular resistance leading to pulmonary artery hypertension (PAHT). At last, PAHT is responsible for right ventricular failure, which may end in death ,2. Patieet al. [et al evidenced a decrease in mean arterial pressure [The effect of etomidate on vascular reactivity has been poorly investigated. Early clinical studies showed that anaesthesia with etomidate induces little or no change in both pulmonary and systemic arterial pressure ,4. Murdaet al.  had obsepressure . In expepressure . Experimpressure . The effPropofol has become a widely used general anaesthetic during the last decade and was studied in many clinical trials. However, its effect on pulmonary vasculature is unclear. In some clinical trial, propofol altered neither pulmonary mean arterial pressure nor pulmonary vascular resistance, though it decreased systemic vascular resistance , whereas2+ homeostasis [versus normoxic rat and human isolated pulmonary arteries. Since chronic hypoxia has differential effects on the systemic versus the pulmonary vasculature, we compared the results obtained in rat pulmonary artery with those obtained in systemic thoracic vasculature, i. e., thoracic aorta, in order to determine if the changes in the effect of anaesthetics observed in pulmonary artery were specific.Most of the experimental studies have been performed on normal arteries. However, PAHT is associated with morphological and functional changes of pulmonary artery, mainly hypertrophy and hyperplasia of smooth muscle cells, and altered vascular reactivity and Caeostasis ,16. Theseostasis -22. We hThe animals used in this study were treated and sacrificed in accordance with national guidelines, and the protocol accepted by local animal experimentation committee. Male Wistar rats, 8–10 weeks old, were exposed to a simulated altitude of 5500 m (barometric pressure 380 mmHg) in a well-ventilated, temperature-controlled hypobaric chamber for 14 days, as previously described ,24. SuchRat pulmonary arteries and aorta from normoxic and hypoxic animals were obtained as follows: for each experiment, a rat was killed by cervical dislocation. The heart and lungs were removed en-bloc, and the extrapulmonary artery and the thoracic part of the aorta were rapidly dissected out. From each specimen, 3 rings 3–4 mm in length were obtained from the main, left and right extrapulmonary artery, and 3 rings of similar length from the thoracic portion of the aorta.1) was 84.4 ± 6.4% of predicted values and mean partial O2 pressure was 83.7 ± 3.3 mm Hg. Quickly after resection, segments of pulmonary arteries  were carefully dissected from a macroscopically tumour-free part of each of the histological pieces and transferred to the laboratory in an ice-cold physiological saline solution. Segments were then cut into rings measuring about 4–5 mm in length for isometric contraction measurements. Use of human tissues was performed according to national guidelines, in compliance with the Helsinki Declaration. Since tissues were obtained incidentally to patient surgery and discarded by the histological pathologist, specific ethical approval of the protocol was not required by French laws.Human bronchial rings were obtained from lung pieces collected for histological examination following resection for carcinoma. Specimens were selected from 7 patients for whom no sign of pulmonary hypertension appeared after pulmonary radiology and clinical examination. Patients were 60.6 ± 4.1 years old. Their lung function was within the normal range: mean forced expiratory volume in 1 second (FEV2-5% CO2 gas mixture. The upper stainless clip was connected to an isometric force transducer (EMKA Technologies). Tissues were set at optimal length (Lo) by equilibration against a passive load of 1.5 g in rat aorta, normoxic human and rat pulmonary arteries, and 2.5 g in CH rat pulmonary arteries, as determined for these types of preparation in control experiments (data not shown).Isometric tension was measured in intact, i. e., with endothelium, vessel rings that were mounted between two stainless steel clips in vertical 20 ml organ baths of a computerized isolated organ bath system  previously described ,24. Bath-6 M phenylephrine (PHE). According to the Nernst equation, 100 mM KCl depolarises the membrane potential close to -10 mV, which opens the voltage-operated Ca2+ channels and thus activates the electromechanical coupling. PHE is an α1-adrenergic agonist that binds to G protein-coupled receptor and acts mainly via InsP3 production and Ca2+ release from intracellular stores, the so-called pharmacomechanical coupling.10-6 M PHE induces an inframaximal contractile response, as determined from a cumulative-concentration response curve to PHE in rat aorta and pulmonary artery . Upon KCl or PHE administration, when the maximal contraction was obtained, propofol or etomidate was added to the vessel rings in cumulative half-log increments from 10-6 to 10-3 M. The ring tension was measured when the response stabilized, i.e., after an equilibration time about 15 min, and expressed as a percentage of the maximal initial contraction of that ring. To avoid any bias due to time-dependent change in tension, the anaesthetic-induced relaxation was normalized to a paired temporal ring, i.e., a ring experimented simultaneously in similar conditions but without exposure to the anaesthetics.The relaxant effect of cumulative concentrations of etomidate and propofol on precontraction to phenylephrine and KCl was assessed as follows. At the beginning of each experiment, prior to exposure to anaesthetics, a contraction was elicited by either a hyperpotassic extracellular solution containing 100 mM KCl or 10®, Zeneca laboratories, Cergy France) and etomidate  were obtained from their clinically used presentations. We verified that the vehicle of each of the drugs had no effect per se on contractile responses up to the maximal concentration used in the present experiments, i. e., 1.65%. Normal KH solution contained (in mM): 118.4 NaCl, 4.7 KCl, 2.5 CaCl2.2H2O, 1.2 MgSO4.7H2O, 1.2 KH2PO4, 25.0 NaHCO3, 11.1 D-glucose, pH 7.4. For KCl-induced contraction, KCl was substituted to NaCl for the desired concentrations, in order to keep the osmotic pressure constant.PHE were purchased from Sigma . Propofol (Diprivan50) and by 30% (IC30), reported as negative logarithm , according to Lovren and Triggle [max refers to the maximal relaxation obtained at the maximal anaesthetic concentration (10-3 M).The relaxation induced by each concentration of anaesthetic was expressed as a percentage of the contractile response of the paired temporal control. The concentration-dependent relaxation curves were then fitted by a non-linear Boltzman equation used to determine the concentrations of anaesthetics that reduced the maximal contraction by 50% , our results suggest that the effect of these anaesthetics on haemodynamics may be greater in patients suffering chronic hypoxia, especially on pulmonary haemodynamics. CH has different effects on systemic and pulmonary vasculature. It has been shown that hypoxia induces systemic vasorelaxation and CH does not induce morphological changes in systemic vasculature . By contThe enhanced effect of the anaesthetics was mainly observed on PHE-precontracted PA. Hence, the increased sensitivity of CH PA is mainly due to an enhanced effect of the anaesthetics on the pharmacomechanical coupling. This suggests that chronic hypoxia modifies the pharmacomechanical coupling of pulmonary artery, in agreement with previous findings on CH-induced calcium signalling in pulmonary arterial smooth muscle cells . Since t2 and 5% CO2. Since anesthetized patients are usually ventilated with hyperoxic gas mixture, our experimental conditions remain, nevertheless, clinically relevant.Though pulmonary artery and aorta rings used in this study were from chronically hypoxic animals, the experimental conditions for isometric measurements were not hypoxic, rather hyperoxic, since tissues were bubbled with 95% O-1 and 0.5–1,6 μg.mL-1, respectively [-5-10-4 M for propofol concentration, and about 2–6.10-6 M for etomidate. At these concentrations, below IC30 values, etomidate has no significant relaxant effect in normoxic tissues and, though enhanced, small effect on CH ones. This may explain why clinical studies have generally not concluded to a relaxant effect of etomidate on PA [30 values. This may explain why some authors have concluded that propofol infusion at clinical doses decreases pulmonary arterial pressure and pulmonary arterial resistances [ex vivo data to in vivo conditions, in particular because of the high protein binding of these compounds which decreases their free concentrations and hence their biological effect [Blood concentrations of propofol and etomidate, following clinical injection, are about 3–15 μg.mLectively -41. Thiste on PA , and sugl effect .In conclusion, our study demonstrates that in normoxic rats etomidate and propofol have a relaxant effect on pulmonary artery, acting mainly on the electromechanical coupling and, to a lesser degree, on the pharmacological coupling. A relaxant effect was also observed in human pulmonary artery, though human PA appears to be less sensitive to the anaesthetics than rat one. The effects of both anaesthetics were greater on PA from an animal model of hypoxia-induced pulmonary hypertension. This enhanced relaxant effect was specific to PA and was mainly seen on the pharmacomechanical coupling. Etomidate appears to be more efficient than propofol at identical concentration. However, comparison that take into account difference between etomidate and propofol concentrations used at clinical doses indicates that etomidate is less potent than propofol, which may have an effect on pulmonary haemodynamics, especially in subjects suffering CH and PAHT. Although these findings provide experimental support for the preferential use of etomidate in patients suffering from PAHT, the clinical relevance of the observations requires further investigation.CCRC: cumulative-concentration response curveHC: chronic hypoxia50: concentration that reduces the maximal contraction by 50%IC30: concentration that reduces the maximal contraction by 30%ICKH: Krebs-HenseleitPA: pulmonary arteryPHE: phenylephrine50: negative logarithm of IC50pIC30: negative logarithm of IC30pICmax: maximal apparent relaxationRThe author(s) declare that they have no competing interests.NO participated in the conception of the study and its design, the experiments in rat and human tissues, participated in the analysis of the data, carried out the statistical analysis, and helped the draft of the manuscript. BM participated in the experiments in rat tissues and the analysis of data. HC participated in the contractile experiments in rat and human tissues. RM participated in the design of the study and helped the draft of the manuscript. ER participated in the conception of the study and its design, helped in statistical analysis and drafted the manuscript.The pre-publication history for this paper can be accessed here:


Mycobacterium tuberculosis directly from clinical specimens have appeared in the world literature since this technology was first introduced. NAA tests come as either commercial kits or as tests designed by the reporting investigators themselves (in-house tests). In-house tests vary widely in their accuracy, and factors that contribute to heterogeneity in test accuracy are not well characterized. Here, we used meta-analytical methods, including meta-regression, to identify factors related to study design and assay protocols that affect test accuracy in order to identify those factors associated with high estimates of accuracy.More than 200 studies related to nucleic acid amplification (NAA) tests to detect M. tuberculosis in sputum samples. Sources of heterogeneity in test accuracy estimates were determined by subgroup and meta-regression analyses. Among 84 studies analyzed, the sensitivity and specificity estimates varied widely; sensitivity varied from 9.4% to 100%, and specificity estimates ranged from 5.6% to 100%. In the meta-regression analysis, the use of IS6110 as a target, and the use of nested PCR methods appeared to be significantly associated with higher diagnostic accuracy.By searching multiple databases and sources, we identified 2520 potentially relevant citations, and analyzed 84 separate studies from 65 publications that dealt with in-house NAA tests to detect IS6110 as an amplification target, and the use of nested PCR methods appeared to be associated with higher diagnostic accuracy. However, the substantial heterogeneity in both sensitivity and specificity of the in-house NAA tests rendered clinically useful estimates of test accuracy difficult. Future development of NAA-based tests to detect M. tuberculosis from sputum specimens should take into consideration these findings in improving accuracy of in-house NAA tests.Estimates of accuracy of in-house NAA tests for tuberculosis are highly heterogeneous. The use of  Accurate and early diagnosis of tuberculosis (TB) is a critical step in the management and control of TB. Because conventional tests for TB have several limitations, nucleic acid amplification (NAA) tests have emerged as promising alternatives. The polymerase chain reaction (PCR) is the best-known and most widely used NAA test. NAA tests are categorized as commercial (kit-based) or in-house ("home-brew"). In-house tests are those assays where the investigators design their own protocols. In-house tests are commonly used in developing countries where commercial kits may not be affordable. The accuracy of NAA tests for TB has been extensively studied since the early 1990s. Since hundreds of studies have evaluated the accuracy of NAA tests, it is now possible to evaluate their overall performance using meta-analysis methods and determine which study design or test-related characteristics are associated with higher diagnostic accuracy.Several systematic reviews and meta-analyses have been published in the past few years on the accuracy of NAA tests for pulmonary and extra-pulmonary TB ,5. BecauWhy do studies on in-house PCR assays produce such highly variable estimates of sensitivity and specificity? Is the variability due to differences in study design or to differences in assay characteristics and laboratory techniques? Are there specific study design features and assay characteristics that yield higher estimates of accuracy? Answers to these questions might help to identify features of NAA tests that maximize accuracy. However, these questions are difficult to address in individual studies. Meta-analytic methods, on the other hand, are well suited to explore the issue of why studies produce variable results. By synthesizing data from multiple studies and increasing the power of analyses, meta-analyses are able to employ techniques that help to identify sources of heterogeneity in study findings. In this meta-analysis, we reviewed 65 published studies on in-house NAA tests for pulmonary TB. The main objective of our meta-analysis was to determine factors associated with heterogeneity as well as higher accuracy estimates of accuracy in studies that evaluated in-house PCR for the diagnosis of pulmonary TB.By searching multiple databases and sources we identified 2520 potentially relevant citations on NAA tests for tuberculosis. After screening titles and abstracts, 434 English and Spanish articles were selected for full-text review and 129 articles reported inclusion of sputum specimens tested by an in-house PCR assay. Sixty-one articles were then excluded mainly because data were not separately provided for sputum samples . Also, three articles were excluded because real time PCR was used and the number was insufficient to place them in a separate category. A total of 65 articles -74, wereIS6110 as amplification target by itself or in combination with other targets, and 30 studies (35%) used other targets . The studies were categorized as those using any chemical method for DNA extraction (including phenol-chloroform) and those in which any physical or mechanical extraction method was used. Sixty-eight of 84 (81%) studies reported a simple PCR protocol (including multiplex PCR), whereas 16 (19%) studies used a nested or seminested PCR protocol. Lastly, 49 of 84 (58%) studies used UV transillumination of an electrophoretic gel, and 35 (42%) used DNA hybridization probes to detect the amplification products.The summary characteristics of the included studies are shown in Table When all 84 studies were evaluated together, the sensitivity estimates ranged from 9.4% to 100%, and specificity estimates ranged from 5.6% to 100%. Both measures were highly heterogeneous (P < 0.001 for test of heterogeneity). Figure IS6110 target in comparison with those using any other target for amplification showed a DOR 1.7 times higher than studies that used other targets. A similar result was obtained when studies that used UV transillumination of a gel were compared to those that used a probe for detection. Studies that used chemical reagents for DNA extraction produced DOR estimates that were about 1.12 times greater than studies that used physical methods, indicating that the use of chemicals (including phenol chloroform) does not significantly improve the test accuracy. Only five studies reported the analysis on smear negative samples. When stratified by smear status, no major difference was seen in the DOR. But this result may be due to the small number of studies reporting only smear negative samples for diagnosis.In order to identify factors associated with heterogeneity, we performed stratified (subgroup) analyses. Table As in our previous meta-analyses ,3, none IS6110 as target of amplification, and studies that used nested PCR methods produced RDOR that were significantly higher than those that used other amplification targets or PCR methods. We present the SROC curves for these subgroups in figures 3A and 3B for target and amplification technique, respectively, to show the trade off between sensitivity and specificity. Blinding, detection technique and smear status showed a slightly higher RDOR but they were not statistically significant in the final regression model. Chemical-based DNA extraction did not produce a significant RDOR, indicating that the use of any chemical reagent for DNA extraction did not substantially affect diagnostic accuracy. No difference was seen in DOR in those studies that used phenol-chloroform versus any other DNA extraction method (data not shown).As shown in Table M. tuberculosis in sputum samples showed a summary receiver operator characteristic (SROC) of 97%, indicating an overall high accuracy of these tests. However, because of significant heterogeneity in sensitivity and specificity estimates, clinically meaningful estimates of accuracy could not be derived. Our analysis showed substantial variability in specificity and sensitivity estimates, and it is clear that in-house PCR tests produce highly inconsistent estimates of diagnostic accuracy. Heterogeneity was clearly evident in the results and could not been explained fully even after stratified analyses. Variability in study design, study quality, and differences in thresholds (cut-points) across studies might account for some of the observed heterogeneity. Nevertheless, the meta-regression analysis highlighted some variables that do appear to yield higher accuracy estimates. The use of IS6110 as the amplification target, and the use of a nested PCR protocol appear to enhance accuracy. It is therefore worth considering the inclusion of these elements in in-house PCR protocols. Our analyses also suggest that the methods used for DNA extraction and signal detection were not critical.Diagnostic methods and, therefore, control of tuberculosis would be greatly improved by the standardization and application of nucleic acid amplification tests. Our meta-analytical review of 84 in-house PCR-based studies to detect IS6110 target sequence in the protocol, and the use of nested PCR methods appear to significantly increase the diagnostic accuracy of PCR. In our previous meta-analysis on NAA test for tuberculous pleuritis, we found that NAA tests that used IS6110 targets produced DOR estimates 2.5 times higher than tests that used other target sequences  / [(1 - specificity)/specificity]DOR = Positive likelihood ratio / Negative likelihood ratioa priori as potential sources of variability: study design, blinded interpretation of NAA test and reference standard, type of PCR test, target sequence amplified, use of probes to detect amplification products, and use of phenol-chloroform for DNA extraction. The results of the meta-regression model are expressed as relative diagnostic odds ratios (RDOR) [Each study in the meta-analysis contributed a pair of numbers: TPR and FPR. Since these measures are correlated and vary with the thresholds (cut points for determining test positives) employed in the individual studies, it is customary to analyze TPR and FPR proportions as pairs, and to also explore the effect of threshold on study results . We summs (RDOR) ,9. An RDLLF designed the study, carried out the study selection, data abstraction, analysis and drafted the manuscript. MP participated in study selection, checked data abstraction and analysis. JC participated in the design of the study and critically reviewed the manuscript. LWR provided input in study design and critically reviewed the manuscript.