This work aims to explore the effects of adaptive statistical iterative reconstruction (ASiR) and model-based iterative reconstruction (MBIR) algorithms in reducing computed tomography (CT) radiation dosages in abdominal imaging.CT scans on a standard male phantom were performed at different tube currents. Images at the different tube currents were reconstructed with the filtered back-projection (FBP), 50% ASiR and MBIR algorithms and compared. The CT value, image noise and contrast-to-noise ratios (CNRs) of the reconstructed abdominal images were measured. Volumetric CT dose indexes (CTDIvol) were recorded.At different tube currents, 50% ASiR and MBIR significantly reduced image noise and increased the CNR when compared with FBP. The minimal tube current values required by FBP, 50% ASiR, and MBIR to achieve acceptable image quality using this phantom were 200, 140, and 80 mA, respectively. At the identical image quality, 50% ASiR and MBIR reduced the radiation dose by 35.9% and 59.9% respectively when compared with FBP.Advanced iterative reconstruction techniques are able to reduce image noise and increase image CNRs. Compared with FBP, 50% ASiR and MBIR reduced radiation doses by 35.9% and 59.9%, respectively. CT scan has been extensively applied in clinical practice and is currently an indispensable tool for various clinical purposes. Its usage has been steadily increased annually. In United State alone, CT scan has been increased from estimated 1.3×10The International Commission on Radiation Protection has suggested that CT scanning practice should keep the radiation dose at the lowest achievable level to minimize the radiation exposure Abdomen is a common place for CT scans, from where a large range of multi-phase scan is routinely performed. In addition, multiple organs are located inside the abdomen, and each comes with diverse density, shape and components, they added up the complexity and requirement of high quality image for clinical evaluation and diagnosis. Previously strategies to achieve low-dose scans primarily include automatic tube current modulation, tube voltage reduction, pitch augmentation, and scan range control A biomedical RANDO standard male phantom for radiotherapy with a height of 175 cm and a body weight of 73.5 kg  was used . The abdThe abdomen of the phantom was scanned repeatedly by a high-definition CT scanner  and then processed on an advanced image processing station . The tube currents were set at 400, 350, 300, 250, 200, 180, 160, 140, 120, 100, 80, 60, 50, 40, 30, 20, and 10 mA. Other parameters included a tube voltage of 120 kV, X-ray tube rotation time of 0.60 s, a pitch of 0.984, a slice thickness of 5 mm, an interlayer thickness of 5 mm, a matrix of 512×512, and a DFOV of 35 cm. The volumetric CT dose index (CTDIvol) at the different tube currents were recorded after each scan.2. The copy and paste functions were used to propagate ROI into different sets of images to ensure the consistency and repeatability. Corresponding CT values, image noise (SD), and contrast-to-noise ratios (CNRs) were recorded. The CNR was calculated based on the following formula: CNR = ACT is the CT value of the iodinated solution inside a test tube on the measured bedding plane, BCT and BSD are the CT value and standard deviation of the background abdominal soft tissue. We also defined the noise decrease rate and CNR increase rate for the two iterative reconstruction (IR) algorithms using the values of FBP and reference standard: SD decrease rate (IR) = (SDFBP-SDIR)/SDFBP×100%;CNR increase rate (IR) = (CNRIR-CNRFBP)/CNRFBP×100%.The images obtained at the different tube currents were reconstructed using three different algorithms; FBP, 50% adaptive statistical iterative reconstruction (ASiR), and model-based iterative reconstruction (MBIR), with a reconstructed slice thickness of 0.625 mm. The reconstructed images were then transferred to the ADW4.5 workstation. An abdominal window with a level of 35 HU and a width of 400 HU was adopted for all the images. Five different bedding planes were selected from each image group for placingfive different regions of interest (ROI). The ROI was circular with an area of approximately 100 mmTo increase the accuracy of the diagnosis, all of the images were evaluated by three senior radiologists who were blinded to the reconstruction algorithms. A 3-point scaling was employed to define the image quality, with score 1 being the best and score 3 the worst. Specifically, score 1 was defined as a clear image in which the circular hole , but no differences were found in the CT values among the different reconstruction models (P>0.05).All of the models presented in this work increased image noise with reduced CNRs as the tube current decreased . At the At the different tube currents (400−10 mA), the 50% ASiR algorithm decreased the image noise by 28%–32% when compared with the noise of FBP; and by MBIR model, the noise decreased by 45%–86%. We also compared the CNRs among three algorithms, the 50% ASiR increased the CNRs by 28%–32% when compared with FBP; and for MBIR, it increased by 46%–84% .According to the subjective assessments, the minimal required diagnostic radiation doses by FBP, 50%ASiR, and MBIR algorithms were 200 mA, 140 mA, and 80 mA, respectively. At the identical image quality, the doses that required by the 50%ASiR and MBIR decreased by 35.9% and 59.9% when compared with that by FBP algorithm.CT radiation dose can be affected by multiple factors, such as tube voltage, tube current, time of exposure, scan range, pitch, X-ray energy, scattered X-ray content, the size of the collimator, structure of the front ray filter, and geometric size of the scanner The principle of the FBP algorithm is based on the assumption that projection data have no image noise, whereas the noise is an innate property of projection data; therefore, the traditional FBP algorithm is subject to noise generation and artifacts. Considering the fact that image noise increases as the tube current decreasing, proper radiation dose is prerequisite to ensure good quality of images By establishing the noise models of the scanned objects, ASiR employs the iterative calculation method and effectively reduce image noise while maintaining image resolution MBIR is an advanced iterative reconstruction algorithms, it uses five models including the three-dimensional optical models and noise model during iterative reconstruction to remove statistical noise and optical fuzzy effect from raw data Furthermore, our results also indicated that FBP image noise was increased as the tube current decreasing. We noted that 50% ASiR decreased the noise by 30% and this noise decrease rate did not depend on the tube current values when it was compared with FBP algorithm. In contrast to 50% ASiR, the noise decreasing rate by MBIR increased gradually as the tube current value decreased, the noise reduction rate was 54% when the conventional radiation dose for an abdominal examination (300 mA) was selected, while when the dose was adjusted to 80 mA, the noise reduction rate increased to 69%.The subjective assessments in this study revealed that the respective minimal tube current values for the interest of diagnosis by FBP, 50% ASiR and MBIR were 200 mA, 140 mA, and 80 mA, respectively. Therefore, MBIR-based reconstructed images had the lowest noise level, but the highest CNR and hence, the lowest radiation dose among all the three algorithms evaluated, indicating that MBIR is a much superior algorithm in reducing the radiation dose over ASiR and FBP.However, our study has some limitations. First, as we used phantom as a model, which only has a simple structure such as soft tissue and bone, rather than human abdominal organs and fat, therefore, the results remain to be confirmed in clinical practice. Second, because MBIR reconstruction model requires large amount of calculation, longer time are expected during the generation of image at X-ray optical system and data collection. The reconstruction of MBIR was about 1 h per case, equaled to 0.09 frame/s. The reconstruction speed of FBP was 15 frames/s, and that of ASiR was 10 frames/s To summarize, both 50% ASiR and MBIR algorithms can significantly decrease the image noise compared with FBP, and MBIR has a better effect in lowing the radiation doses. At the identical image quality, 50% ASiR and MBIR reduce the radiation dose by 36% and 60% over the FBP algorithm.


Chronic inflammation is related to both obesity and diabetes. Our aim was to investigate to what extent this inflammation contributes to the association between obesity and diabetes.Using a case-cohort design, we followed 567 middle-aged individuals who developed diabetes and 554 who did not over 9 years within the ARIC Study. Weighted Cox proportional hazards analyses permitted statistical inference to the entire cohort.2), compared to those with BMI<25 kg/m2, presented a large increased risk of developing diabetes , as did those in the highest (compared to the lowest) quartile of waist circumference , in analyses adjusted for age, gender, ethnicity, study center, and parental history of diabetes. Notably, further adjustment for adiponectin and inflammation markers halved the magnitude of these associations . In similar modeling, attenuation obtained by adding fasting insulin, instead of these markers, was only slightly more pronounced HR[obesity]=2.7, 95%CI 1.7–4.1; and HR[waist]=3.6, 95%CI 2.3–5.8).Obese individuals (BMI≥30 kg/mThe marked decrease in the obesity-diabetes association after taking into account inflammation markers and adipokines indicates their major role in the pathways leading to adult onset of diabetes in obese individuals. Diabetes is a health problem of increasing concern. The World Health Organization estimates that 2.9 million deaths per year are attributable to diabetes, and this number is likely to increase by more than 50% in the next decade. Much ofObesity has been shown to lead to functional and morphological damage to remote tissues via circulating factors-5. HotasThe ARIC study offers a unique opportunity to examine this through a case-cohort study rich in measurements of inflammation markers and adipokines. Thus, we investigated the possible role of inflammatory processes, as indicated by biomarkers, and adipokines in the obesity-diabetes association, by quantifying the degree of reduction in the strength of the obesity-diabetes association when taking these factors into account.Between 1987 and 1989 the Atherosclerosis Risk in Communities (ARIC) study recruited a population-based cohort of 15,792 men and women aged 45–64 years from four US communities. In the present analyses, we used a case-cohort design, as previously described in the investigation of the role of several inflammation biomarkers in the development of diabetes in the ARIC study. Prior t2 were classified as obese, those presenting a BMI value equal or greater than 25 but lower than 30 kg/m2 were classified as overweight, and those with BMI < 25 kg/m2  were considered as having normal body weight. In order to assess the association of waist circumference values with incidence of diabetes, we also compared waist circumference extreme quartiles.We measured waist girth at the umbilical level and hip circumference at the maximum hip girth in order to obtain the waist-to-hip ratio, and divided weight by height squared to obtain the body mass index (BMI). Individuals presenting BMI ≥ 30 kg/mWe defined parental history of diabetes as a report of diabetes in either parent, and hypertension as sitting blood pressure of 140/90 mm Hg or more or use of antihypertension medication.We used a score, rangingWe used weighted Spearman correlations to describe crude associations between BMI and waist circumference and other variables, and weighted ANCOVA to compute adjusted BMI means in diabetes cases and non-cases. We performed Cox proportional hazards regressions to analyze the relation between BMI and time to onset of diabetes, with appropriate weighting for the stratified sample selection. In these analyses, weights are defined as the inverse of the ethnicity-specific sampling fractions. Leptin quartiles were defined in a sex-specific manner. Adipone2), 41% were overweight (25 ≤ BMI < 30 kg/m2) and 28% were obese (BMI ≥ 30 kg/m2) at baseline. BMI mean values were 26.9 (95%CI: 26.4–27.5) for men, 26.8 (95%CI 26.4–27.4) kg/m2 for women, and were higher in African-Americans  than whites . According to the criteria of the Third Report of the National Cholesterol Education Program (NCEP) criteria[Characteristics of cases and non-cases have previously been reported. In briecriteria, 52% of Spearman correlations, assessed in the cohort random sample Table , show th2 ≤ BMI < 30 kg/m2) and waist circumference were the main exposure variables. Although the same pattern was also observed when fasting insulin was the main exposure, the minimally adjusted hazard ratio was somewhat larger and the reduction with adiponectin adjustment, more pronounced. After including the adipokine leptin in these analyses, the association increased slightly, particularly so in the waist circumference analysis. When additionally adjusting for hypertension and other variables associated with the metabolic syndrome, associations were further reduced.Table As insulin resistance is considered a central mechanism in the causation of type 2 diabetes, we performed similar models of progressive adjustment, but adding insulin prior to adiponectin and inflammation markers. As seen in Table No heterogeneity in the pattern of these associations was observed across categories of ethnicity or smoking. A secondary analysis, excluding 27 individuals originally defined as non-cases but with glycemia post 75 g oral glucose load of ≥11.1 mmol/L at ARIC Visit 4, produced similar results (data not shown).2 in models adjusting for age, gender, center, ethnicity, and parental history of diabetes. However, once additional adjustment for adiponectin and inflammation markers was performed, the relative excess risk (1-HR), the measure which best permits comparison of the strength of the main exposure associations following different adjustments, dropped by more than 50%. Similar results were observed when waist circumference and fasting insulin were considered in the models instead of obesity , with changes in adipocyte gene expression and with infiltration of inflammatory cells, most notably macrophages into adiHowever, we and otheBench research supports these results. Endoplasmatic reticulum (ER) is a major site for intracellular protein, lipid and sterol synthesis. In balancing intracellular supply and demand in the process of constructing new molecules, the ER has now been shown to be also a major generator of metabolic and inflammatory signals. ER stress is an early consequence of nutrient excess and a cause for the development of inflammation and insulin resistance-28. ER sThe increased risk for main exposures after addition of leptin to the models merits interpretation. This adipokine has known actions which protect against the development diabetes, but itsThe role of the additional risk factors – hypertension, non-esterified fatty acids, triglycerides and HDL – in diabetes causation, and thus in confounding or mediating associations seen with the above-mentioned factors, is less clear. Hypertension, as mentioned above, could result, in part, from chronic vasoconstriction caused by adipokine-induced, inflammation-mediated endothelial dysfunction, which in parallel leads to insulin resistance and diabetes. Increased production of angiotensinogen by inflamed adipose tissue is another possible mechanism. Within Our study has some limitations. First, the fact that variables that could be considered potential mediators in this analysis  were measured at the same moment as the main exposure (obesity) limits our ability to test hypotheses regarding mediation with more sophisticated techniques. NonetheInsulin resistance, along with beta cells failure, is believed to be a major final common pathway from obesity to type 2 diabetes. The reduction in obesity-diabetes relative risk seen with adjustment for insulin is consistent with this view. The fact that markers of chronic inflammation led to a similar relative risk reduction, suggests a major, if not nearly equivalent role for inflammatory pathways in the causation of type 2 diabetes.Our interpretation of these analyses should not be construed as suggesting a linear pathway (nor multiple linear pathways) from obesity to type 2 diabetes. Undoubtedly the process is a much more complex one, with various processes manifesting along the causal pathways, including, among them, inflammation and insulin resistance/beta cells failure. Given the integration of these processes and the continuum in their installation over time, definition of what comes first is difficult to address epidemiologically and may not be the most relevant question. Population approaches, such as Mendelian randomization, particularly when investigating upstream inflammatory mediators, may complement basic science research in further elucidating the role of chronic inflammation in the progression from obesity to type 2 diabetes. The furOur findings, when coupled with the literature, support a major role of adiponectin and other inflammatory factors in mediating the progression from obesity to type 2 diabetes.AMK: Activated protein kinase; ANCOVA: Analysis of covariance; ARIC: Atherosclerosis Risk in Communities; C3: Complement C3; ER: Endoplasmatic reticulum; ICAM-1: Intercellular Adhesion Molecule-1; NCEP: Third Report of the National Cholesterol Education Program.The authors declare that they have no competing interests.All authors contributed significantly to this paper. VCL, MIS and BBD researched data and wrote the manuscript. JSP and JHY researched data, contributed to the discussion and reviewed/edited the manuscript. DC researched data and reviewed/edited the manuscript. CB reviewed/edited the manuscript. All authors read and approved the final manuscript.


To the Editor:Stenotrophomonas maltophilia has emerged as an important nosocomial pathogen, especially in debilitated and immunocompromised persons .Salads were purchased from reputable supermarkets and transported immediately to the laboratory. Ten grams of salad was homogenized in 90-mL sterile saline in a stomacher for 1 to 2 min. Aliquots (200 μL) of decimal dilutions of the homogenate were plated onto vancomycin-imipenem-amphotericin B (VIA) agar  and incuPseudomonas aeruginosa (NCTC10662) was used as a control. Because disk diffusion is not a reliable method for determining the susceptibility of S. maltophilia to quinolone antimicrobial agents  of 18 salads. Numbers ranged from 1.50 × 102 to 1.96 × 105 CFU/g . All isolates were susceptible to ciprofloxacin, colistin sulfate, minocycline, and trimethoprim-sulfamethoxazole. Eight (89%) were resistant to chloramphenicol; 7 (78%) to piperacillin/tazobactam; 5 (56%) to ceftazidime, and 2 (22%) to gentamicin.S. maltophilia in products of this type, especially if subject to temperature abuse, are unknown, but in the domestic setting, numbers of the bacterium may increase before use.All products examined were labeled "washed and ready to eat," and, thus, consumers would be unlikely to wash these products before consumption. The growth characteristics of S. maltophilia associated with oilseed rape. However, as with most strains of clinical origin, all the isolates tested here remained susceptible to trimethoprim-sulfamethoxazole and minocycline.All of the 9 isolates examined exhibited resistance to >2 of the 8 agents tested, with 2 isolates resistant to 4 compounds and thus had resistance phenotypes similar to those of strains associated with human infection. These findings are in agreement with those of Berg  of 41 patients, which emphasizes that foodstuffs may be a potential source of this bacterium for some patients. This is a preliminary study, however, and further studies are needed, in particular, molecular typing of food and human-associated isolates, to investigate the hypothesis that intestinal carriage of S. maltophilia may follow consumption of contaminated foodstuffs.The importance of tropenia . Recentltropenia , in a pr


Objective: Chronic menorrhagia causes anemia and impairment of life quality. In this study the aim was the screening of bleeding disorders in adolescents and young women with menorrhagia. Materials and Methods: The study was performed prospectively by pediatric hematologists. A form including demographic characteristics of the patients, bleedings other than menorrhagia, familial bleeding history, characteristics of the menorrhagia, and impairment of life quality due to menorrhagia was filled out by the researcher during a face-to-face interview with the patient. A pictorial blood assessment chart was also used for evaluation of blood loss. All patients underwent pelvic ultrasound sonography testing and women also received pelvic examination by gynecologists. Whole blood count, peripheral blood smear, blood group, serum transaminases, urea, creatinine, ferritin, PFA-100, PT, aPTT, INR, TT, fibrinogen, VWF:Ag, VWF:RCo, FVIII, and platelet aggregation assays were performed. Platelet aggregations were studied by lumiaggregometer. Results: Out of 75 patients enrolled, 60 patients completed the study. The mean age was 20.68±10.34 (range: 10-48) years and 65% (n=39) of the patients were younger than 18 years. In 18 (46%) of the adolescents, menorrhagia subsided spontaneously. In 20% (n=12) of the patients, a bleeding disorder was detected . Conclusion: In patients with menorrhagia, at least complete blood count, peripheral smear, aPTT, PT, VWF:Ag, VWF:RCo, FVIII, and fibrinogen assays must be performed. When there is history of nose and gum bleeding, platelet function assay by lumiaggregometer must also be performed. In nearly 50% of adolescents, menorrhagia is dysfunctional and transient. Detailed coagulation assays can be postponed in adolescents if bleeding history other than menorrhagia and/or family history of bleeding and/or parental consanguinity is absent. All subjects with menorrhagia must consult with gynecologists and hematologists.Conflict of interest:None declared. Menorrhagia is defined as a menstrual blood loss of more than 80 mL per menstrual cycle. Its estimated prevalence in healthy women is 9%-14% . MenstruThe ethics committee of Kocaeli University approved the study. The patients’, and for patients younger than 18 years also their legal guardians’, written informed consent was obtained. The study was performed prospectively by pediatric hematologists between June 2009 and December 2010 in adolescents and women of reproductive age during their admission to the gynecology and adult and pediatric hematology outpatient and emergency care units of Kocaeli University Hospital. Physicians of the Gynecology Department were informed about the study and referrals of patients presenting with menorrhagia were requested. The adult hematology unit was also informed about the study and referral of patients presenting with menorrhagia was requested. Patients presenting with menorrhagia were referred to hematology clinics by second level referral centers due to suspected bleeding disorders. The researcher filled out a form including demographic characteristics of the patients, bleedings other than menorrhagia, familial bleeding history, and characteristics of the menorrhagia during a face-to-face interview with the patient. Pictorial blood assessment charts (PBAC) were also distributed for evaluation of blood loss during menstruation. Patients compared the degree of saturation of their sanitary pads and tampons with those depicted on the chart. Patients ticked the pictures throughout menstruation and wrote the number of the tampons and/or pads used every day. Lightly stained pads or tampons obtained a score of 1, moderately stained pads or tampons a score of 5, and soaked pads or tampons a score of 20. The total points were calculated by the researcher. A PBAC total score of greater than 100 was considered a blood loss of more than 80 mL .To evaluate the quality of life during menstrual periods, patients were asked to rate 7 parameters of daily living on a scale of 0-10, with 0 meaning that menses “does not interfere” with that parameter and 10 meaning that menses “completely interferes”. The parameters were patients’ general activity, ability to work and attend school, family activities, and ability to enjoy life, sleep, mood, and overall quality of life. Total points were 0-35 for the mildly affected and 36-70 for the severely affected [3]. All the patients had pelvic ultrasonography in the gynecology outpatient clinic and women also had pelvic examinations to exclude gynecological pathologies. Patients with gynecological pathologies such as myoma uteri or a history of systemic disease such as diabetes mellitus and hypothyroidism were excluded from the study. Blood samples were obtained in the first 5 days of the menstrual cycle and oral contraceptives (OCs) and non-steroidal anti-inflammatory drugs were withdrawn at least 10 days before. Vacutainer tubes with citrate were used and samples reached the local laboratory within 15 min. Aggregation assays were performed within 3 h. Whole blood count, peripheral blood smear, blood group, ferritin, platelet function analyzer 100 (PFA-100), prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), fibrinogen, von Willebrand factor antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo), factor VIII (FVIII), and platelet aggregation assays were performed. Serum transaminases, urea, and creatinine assays were also performed, because uremia impairs platelet functions and liver disease impairs synthesis of the coagulation factors. Other rare coagulation factor assays were performed when PT and/or aPTT were prolonged. Whole blood count was studied with the Celldyn-3700© (Abbot); biochemistry testing with the Aeroset 70© (Abbot); serum ferritin level with the Modular E-170© (Roche); PFA-100 testing with equipment by Dade-Behring©; PT, aPTT, fibrinogen, thrombin time, and VWF:Ag with Star© (Diagnostica Stago); and platelet aggregation testing and VWF:RCo with Chrono-Log Lumiaggregometry. Collagen, ristocetin, adenosine diphosphate (ADP), and epinephrine were used as agonists. The concentrations of agonists were: collagen, 0.5 mg/mL; ristocetin, 1.5 mg/mL; ADP, 1.0 µM/L; and epinephrine, 1.0 µM/L. Collagen and ADP cartridges were used for PFA-100. High PBAC score and/or duration of menses greater than or equal to 7 days with a sensation of flooding or bleeding through a tampon or pad in ≤2 h, history of treatment of anemia, clots greater than 2.5 cm in diameter, family history of diagnosed bleeding disorder, history of excessive bleeding with tooth extraction, delivery or miscarriage or surgery, history of spontaneous nosebleed that persisted for 10 min or needed medical attention, spontaneous gum bleeds, prolonged bleeding from minor wounds, easy bruising with minimal trauma, or similar bleeding symptoms in the family were regarded as clinical factors supporting existence of a bleeding disorder. The following criteria were used for diagnosis of bleeding disorders. Criteria for diagnosis of Glanzmann’s thrombasthenia (GT) were normal PT, aPTT, platelet count, and morphology; no curve with ADP, epinephrine, or collagen; and normal curve with ristocetin in platelet aggregation by lumiaggregometry. Criteria for diagnosis of Bernard-Soulier syndrome (BSS) were low platelet count, large platelets and normal aggregation with physiologic agonists, and no curve with ristocetin. For laboratory diagnosis of VWD, VWF:Ag, VWF:Rco, and FVIII coagulant assays were used. The normal value for these parameters was 50-200 IU/dL. VWF:Ag levels of 30 IU/dL or lower are required for the definite diagnosis of inherited VWD . VWF:Ag is less than 50 IU/dL in most patients with type 2A, 2B, or 2M VWD. VWF:Rco is less than 30 IU/dL in types 1 and 2. Levels of 30 to 50 IU/dL are classified as low VWD. In patients with blood group O, VWF:Ag is 25% lower. In type 1, both VWF:Ag and VWF:Rco are low and VWF:Rco/VWF:Ag is >0.5-0.7. In types 2A, 2B, and 2M, the VWF:Rco level is decreased more than VWF:Ag (VWF:Rco/VWF:Ag is <0.5-0.7). In types 1 and 2, the FVIII level may be low or normal. In type 2N, VWF:Ag and VWF:Rco may be normal or decreased. In type 3, VWF:Ag and VWF:Rco are absent (<3). In type 2N and type 3, FVIII is <30 IU/dL and <10 IU/dL, respectively . A review of 11 studies conducted internationally found 13%  of women with menorrhagia to have VWD . In thes The diagnosis of VWD is complex and the diagnosis of mild forms can be difficult. Persons with levels of 30 to 50 IU/dL may need agents to increase VWF levels during invasive procedures or childbirth. Therefore, it is important to correlate severity and cause of bleeding with results of laboratory tests . The levMenorrhagia has been reported to be present in 51% of women with BSS and 13%-98% of women with GT ,16,17. IThe PFA-100 is easy to perform and a quick test, but is not the gold standard compared to lumiaggregometry. In our study, the number of the patients with bleeding disorders was too low to make a conclusion about the sensitivity and specificity of screening with PFA-100, although closure times seemed longer in patients with bleeding disorders. Eighty-one women with a physicians’ diagnosis of menorrhagia underwent PFA-100 and bleeding time tests to evaluate their effectiveness as screening tools for VWD and platelet dysfunction. Data showed that PFA-100 had a sensitivity of 80%, specificity of 89%, positive predictive value (PPV) of 33%, negative predictive value (NPV) of 98%, and efficiency 88% for VWD. For platelet aggregation defects, the PFA-100 closure time had a sensitivity of 23%, specificity 92%, PPV of 75%, NPV of 52%, and efficiency of 55%. Neither the PFA-100 nor the bleeding time tests are effective for purposes of classifying women for standard platelet aggregometry testing in women presenting with menorrhagia . In a study by Kadir et al., menorrhagia since menarche was noted in 11 (8.9%) of 123 women without a bleeding disorder compared with 13 (65%) of 20 women with VWD (p=0.001) and 4 (66.7%) of 6 women with FXI deficiency (p<0.001) . AlthougIt is clear that in the absence of iron-deficiency anemia, heavy menstrual bleeding is a subjective complaint and up to 50% of women describing menorrhagia will have a measured monthly blood loss within normal limits . The PBAMedical treatments including hormones, antifibrinolytics, and iron salts are useful in the management of patients without gynecological pathology regardless of the etiology. The concentrated spray form of desmopressin is not available in Turkey and the parenteral form can be used only in inpatient settings. Consequently, we did not try desmopressin (DDAVP) in patients with VWD. We used oral tranexamic acid in all patients and it reduced menstrual blood loss. Oral tranexamic acid  for 5-7 days of the menstrual cycle was our practice. In some studies, a reduction in menstrual blood loss by 50% was shown . It can Unexplained hemorrhage often triggers surgical interventions such as endometrial ablation and/or hysterectomy. In our study, only one surgical intervention, splenectomy for chronic ITP, was performed. In adolescents and women of child-bearing age, preservation of fertility is essential. The limitations of this study were the low number of patients and the lack of routine screening of rare coagulation defects  when PT, PTT, and TT were normal. FIX carriage was also not screened when there was no history of hemophilia B in the family. Multimer analysis of VWF was also not available. In addition, some assays were missing in some patients due to subjects’ incompliance to the study. We tried to compare some characteristics of the patients with and without underlying bleeding disorders to find predictive factors for underlying bleeding disorders. However, the statistical power was low due to patient numbers in the groups. In conclusion, screening of subjects with menorrhagia for underlying bleeding disorders is laborious and not all coagulation assays are available in many routine laboratories. In patients with menorrhagia, at least complete blood count, peripheral smear, aPTT, PT, VWF:Ag, VWF:RCo, and fibrinogen assays must be performed. When there is a history of nose and gum bleeding, platelet function assay by lumiaggregometer must also be performed. In patients with a history of surgical bleeding, screening of rare coagulation deficiencies is also required. In nearly 50% of adolescents, menorrhagia subsides with the maturation of the hypothalamic–pituitary–ovarian axis. Detailed coagulation assays can be postponed in adolescents if bleeding history other than menorrhagia and/or family history of bleeding and/or parental consanguinity is absent. All subjects with menorrhagia must consult with gynecologists and hematologists. In subjects with bleeding disorders, future follow-up for management of pregnancy and delivery is essential for the safety of the mother and offspring.Thanks to laboratory technician İlknur Çağlar for her meticulous work in all of the coagulation assays. Thanks also to the physicians that referred patients to us and performed gynecological evaluations. The Kocaeli University Research Foundation sponsored the study. There was no conflict of interest in this study.


The reversibility of hepatic histological damage after restoring bile flow in a murine model was assessed. 25 male Balb C mice  were divided into 5 groups and their common bile duct (CBD) fastened to obstruct the release of gall bladder and liver contents. Group I, CBD untied at day 10, group II at day 15, and groups III and IV at days 20 and 30, respectively. Hematoxilin-eosin stained liver slices were analysed 0, 5, 10 and 20 days after restoring bile flow. Group I showed slight histological lesions (second stage), as cholangiolar bile pigment concretion, pericholangiolar and portal collagen accumulation; group II, mild lesions (third stage), as cholangiolar hamartomatous proliferation and bile duct portal fibrosis; group III showed severe lesions (fourth stage), as loss of functional parenchyma, and also the second and first stage lesions. Group IV died before 30 days. First stage corresponds to absent lesions (control group). Group I recovered totally, group II recovered only from slight lesions and group III had irreversible damage. Severity of lesions increased gradually and accumulatively, irreversible hepatic damage was achieved at 20 days and is deadly at 30 days. Our model of temporary CBD obstruction was suitable to assess reversibility of hepatic histological damage. Obstruction of the common bile duct  is known to occur as part of the physiopathology of choledocholithiasis or physical compression by neoplasia or inflammation of nearby organs. This obstruction may disturb the normal hepatic morphology, by increasing the normal pressure inside liver lobules, leading to minor lesions as centrolobular cholestasis, or even more severe damage, as hepatic cirrhosis , 2. HoweSeverity of lesions increase as the obstruction persists, leading to the appearance of histopathological disorders such as sinusoidal congestion in the early stages and biliary fibrosis with cholangiolar hamartomatous proliferation when obstruction persists even more; these disorders were confirmed with models reported elsewhere . This moTherefore, the aim of this study is to determine the reversibility of hepatic histological damage induced by temporary surgical obstruction of the common bile duct in Balb C mice, as a model of bile flow obstruction in man.25 male Balb C mice, age 6 weeks, weighing 25 to 35 g, bred in laboratory, were used in this study. Animals were divided into 4 groups including 5 animals each, plus a control group with 5 animals having intact CBD, maintained in 36 × 26 × 16 cm acrylic cages under controlled conditions, temperature 21 ± 3°C, 55 ± 10% relative humidity, and 10-12 air changes per hour, with cycles of light/darkness of 12/12 hours and free access to water and food, pine shavings as supporting material. Animals were free of viruses and other pathogens. The research project was approved by the Ethical Committee for the Care of Laboratory Animals (ECCLA) of our Institution.After being completely anaesthetised with sodium phenobarbital (5.5 mg/kg i.p.), a surgical 1.5 cm length incision was made to all the animals, in the right upper quadrant, getting through the skin, then subcutaneous tissue (ST), superficial and profound aponeurosis, traverse and rectus muscles and peritoneum. A small polyethylenecellulose canule, 3 mm internal diameter and 8 mm length, was inserted into the gallbladder, in order to allow the total recovery of bile flow into the small intestine when untying the CBD. The incision was closed according to two plains, the first with peritoneum, muscles and aponeurotic fascia and, the second, with skin and subcutaneous tissue.Surgical wounds were cleaned with sterile soap, water and hydrogen peroxide and the animals were put back into their cages directly after the surgery, and heat was given to them with an electric lamp. The first day, bile duct obstruction was carried out to the four experimental groups, except by the control, and the following experimental scheme was conducted: Day 10: the bile duct of group I was untied and liver biopsies were taken. Day 15, the bile duct of group II was untied and liver biopsies from groups I and II were taken. Day 20, group III CBD was untied and liver biopsies were taken from groups I, II and III. Day 30, the common bile ducts from the remaining 5 mice (group IV) were untied and hepatic biopsies were taken from the whole groups.th day, biopsies from several organs, such as liver, brain, kidneys and lungs were taken from each mouse, and analysed microscopically. Organs were immersed in formalin (10% v/v methanol) during 48 hrs at least, included in paraffin, sliced into 2 to 8 μm portions, stained with haematoxylin-eosin and photomicrographed. Two pathologists carried out microscopic analyses independently, in a double-blinded manner, and their results were confronted. Severity of the histological damage was categorised into four stages or grades: First, null or absent lesions. Second, slight lesions, including bile pigment concretion and collagen accumulation. Third, mild lesions, including hamartomatous ones. Fourth, severe lesions, including loss of functional parenchyma.At the 30p≤0.05).Data corresponding to the stage of histological disturbance among groups were scored in analogous scale with arbitrary units, and numeric values were assigned to each stage. Data were analysed by the ANOVA test, and the significant differences were evaluated by Tukey test , the entire group I showed an adequate recovery, and the results rendered by both analysts correlated as they observed a noticeable recovery of the lesions 5 days after removing the obstruction. However, such recovery becomes more evident at the 20th day, since the entire group showed no significant histological disturbances. Therefore, special attention was paid to this group, considering the evolution of the lesions found during the 10 day-lasting obstruction and the histological changes that could be observed after bile duct liberation, as seen in previous reports  of hepatic histological disturbances after common bile duct obstruction, and 5 days after removing the obstruction (15 reports , 13.Helpfulness of the temporary common bile duct obstruction procedure is worth to be highlighted, not just for their feasibility in similar studies, but also in the treatment and outcome evaluations, as has been widely described by other authors, both in murine models , 15 or iHepatic histological disturbances such as cholangiolar bile pigment concretion, as well as a slight pericholangiolar and portal collagen accumulation, derived by temporary bile duct obstruction can be reverted. However, further studies are needed to conclude about the total impact of CBD obstruction and hepatic damage.


Salicylic acid (SA) is a naturally occurring phenolic compound. SA plays an important role in the regulation of plant growth, development, ripening, and defense responses. The role of SA in the plant–pathogen relationship has been extensively investigated. In addition to defense responses, SA plays an important role in the response to abiotic stresses, including drought, low temperature, and salinity stresses. It has been suggested that SA has great agronomic potential to improve the stress tolerance of agriculturally important crops. However, the utility of SA is dependent on the concentration of the applied SA, the mode of application, and the state of the plants . Generally, low concentrations of applied SA alleviate the sensitivity to abiotic stresses, and high concentrations of applied induce high levels of oxidative stress, leading to a decreased tolerance to abiotic stresses. In this article, the effects of SA on the water stress responses and regulation of stomatal closure are reviewed. Salix, which is the Latin name for the willow tree . Salicin, the glucoside of salicylic alcohol, was first isolated in 1826 from willow bark, and a large amount of the substance was successfully isolated in 1828. Salicin was then converted into a sugar and an aromatic compound that, upon oxidation, becomes SA. SA, a 2-hydroxybenzoic acid , has a colorless crystalline structure and is widely used in organic synthesis, including the synthesis of aspirin, also known as acetylsalicylic acid. Plants generally contain a few micrograms of SA or less per gram of fresh weight  is involved in the regulation of pathogenesis-related protein expression, leading to plant defense against biotrophic pathogens . It alsoh weight , either Figure 1). These pathways begin with chorismic acid, which is the end product of the shikimate pathway and is synthesized in the plastid. The major pathway is the IC pathway in Arabidopsis thaliana, Nicotiana benthamiana, tomato, and other plants  pathway and the phenylalanine ammonia-lyase (PAL) pathway . PAL, the first enzyme in this pathway, deaminates phenylalanine, leading to the production of trans-cinnamic acid. Trans-cinnamic acid is a precursor for the biosynthesis of diverse phenolic compounds, including lignin, lignans, flavonoids, volatile benzenoid esters, and benzoyl glucosinolates  may catalyze the conversion of IC to SA, given that some bacteria, such as tain IPL . Howeverinolates . Thus, Pacid BA; .NPR1) could be an SA receptor candidate because npr1 mutant plants exhibit a complete lack of resistance against biotrophic and hemibiotrophic pathogens , which includes global transcriptional reprogramming and immune responses to a broad spectrum of pathogens . Previouathogens . Further control . NPR1 po control . AlthougRecently, the NPR1 paralogs NPR3 and NPR4 were identified as SA receptors that bind specifically to SA with different affinities . Both of2 and transpiration. During water deficits, the stomata are closed to slow transpiration and conserve water in the plant, thereby decreasing the CO2 supply and leading to a reduction in photosynthesis. Stomatal opening or closure is achieved by the osmotic swelling or shrinking of guard cells, respectively , which is induced by SA . The exo closure . Two maj closure , and ano closure . In addi closure  and oxal closure . Genetic closure . Howevertivation .Arabidopsis leaves, stomatal closure is induced within 1 h  chloride, an inhibitor of NAD(P)H oxidase  elicitor or to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000, respectively  or SA-deficient (sid2) mutants revealed that 27 genes in two clusters are up-regulated by SA, drought, and the SA-accumulating mutants siz1 and cpr5. Among these genes, 9 are highly expressed in guard cells  was alleviated by methyl SA and methyl jasmonate (JA) vapors (AOX) gene induced by methyl SA and methyl JA vapors  vapors . This reA vapors . The expA vapors , and theg stress . These og stress . Potatoeg stress . The appg stress . When tog stress . The hydg stress . SA treag stress . The appg stress .Arabidopsis SA over-accumulating mutants, such as acd6, cpr5, and siz1  enhanced the accumulation of SA and the freezing sensitivity  to mung bean  reduced the inhibitory effect of high salinity, while higher concentrations of SA (>100 μM) enhanced this effect  to hydroponic solutions containing media and polyethylene glycol (PEG) alleviated the harmful effects of osmotic stress on wheat seedlings . Exogeno biomass . The Ara closure .Arabidopsis and tobacco, treatment with 0.1–0.5 mM SA is optimal for eliciting the highest level of stress tolerance , which induces oxidative burst and cell death  induces high levels of ROS accumulation, leading to a decrease in the capacity to scavenge ROS . The ovetalase 2 , is alle in ICS1 . This restresses . Stress-ll death . Under all death . High coP levels . IntraceArabidopsis  cascade may be involved in the transmission of the SA/ROS signal to regulate downstream genes . Severaln kinase  and wounn kinase , respectis roots .Arabidopsis NPK1-like protein kinase 1, is activated by H2O2, leading to the phosphorylation of MPK3/MPK6 in Arabidopsis plants (2O2 accumulates in mekk1 and mpk4 mutants and activates MEKK1 in protoplasts. Because the MEKK1 protein level is also increased by H2O2(Genetically, the MEKK1-MKK1/2-MPK4 cascade plays a negative role in the regulation of defense responses because the loss of function of either MEKK1 or MPK4 prompts the accumulation of SA . Similars plants . The MEKs plants . H2O2 acd by H2O2, the MEKSalicylic acid plays an important role in the regulation of the abiotic stress responses described above. Application of the appropriate concentration of SA enhances tolerance to abiotic stresses, thereby not only mitigating the damaging effects of abiotic stress tolerance but also enhancing biotic stress tolerance. The important characteristic of SA application is the concentration of applied SA and the method of application, such as foliar spray and hydroponic culture. These methods depend on the plant species; therefore, contradictory results can be reported. Generally, low concentrations or the transient application of SA promotes plant tolerance to abiotic stresses, and high concentrations or the continual application of SA induce inhibitory effects on plant growth and reduce tolerance. It is clear that SA is a very promising compound for the reduction of the abiotic stress sensitivity of numerous plant species.It remains unclear how SA plays a specific role in abiotic stresses. The accumulation of endogenous SA is induced by several abiotic and biotic stresses. However, how the accumulation of SA is distinguished by each stress is not understood. If the mechanism of how plants distinguish the induction of SA by each stress is understood, this knowledge would contribute to the clarification of the specificity of plant responses to abiotic stresses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


GSTP1, APC and RASSF1 have demonstrated involvement with prostate cancer, with the latter two genes playing prominent roles in the field effect. The epigenetic states of these genes can be used to assess the likelihood of cancer presence or absence.PSA-directed prostate cancer screening leads to a high rate of false positive identifications and an unnecessary biopsy burden. Epigenetic biomarkers have proven useful, exhibiting frequent and abundant inactivation of tumor suppressor genes through such mechanisms. An epigenetic, multiplex PCR test for prostate cancer diagnosis could provide physicians with better tools to help their patients. Biomarkers like GSTP1, APC and RASSF1 genes, using methylation specific PCR (MSP). The effect of prostate needle core biopsy sample volume and age of formalin-fixed paraffin-embedded (FFPE) samples was evaluated on an independent follow-up cohort of 51 cancer-positive patients. Multiplexing affects copy number calculations in a consistent way per assay. Methylation ratios are therefore altered compared to the respective singleplex assays, but the correlation with patient outcome remains equivalent. In addition, tissue-biopsy samples as small as 20 μm can be used to detect methylation in a reliable manner. The age of FFPE-samples does have a negative impact on DNA quality and quantity.An initial test cohort of 30 prostate cancer-positive samples and 12 cancer-negative samples was used as basis for the development and optimization of an epigenetic multiplex assay based on the The developed multiplex assay appears functionally similar to individual singleplex assays, with the benefit of lower tissue requirements, lower cost and decreased signal variation. This assay can be applied to small biopsy specimens, down to 20 microns, widening clinical applicability. Increasing the sample volume can compensate the loss of DNA quality and quantity in older samples. In the USA, over 70% of the annually ~1,000,000 performed biopsies for prostate cancer suspicion result in a cancer-free diagnosis , with scFrom the diagnostic perspective, creating a robust, accurate assay is of paramount importance. In addition, from the clinical point of view, the assay should be able to test readily available samples with a range of quantity and quality. When a diagnostic assay consists of several subtests, e.g. a panel of several biomarkers, these can, potentially, be run together in a multiplexed fashion. Although a multiplex assay minimizes sample volume requirements and specimen handling, and the associated operator-related and technical errors, it is important to verify that the generated output reaches the same standards of accuracy and reliability, compared to the individual singleplex biomarker versions.GSTP1APC and RASSF1[ACTB, used to quantitate amplifiable DNA.The current study evaluates a multiplex assay for prostate cancer detection. To reduce the number of false positives and subsequent unnecessary (repeat) biopsies due to a lack of PSA-specificity and spurious extraction of biopsies cores -17, a cond RASSF1, in addiACTB was used as a rough estimate of input DNA quantity and quality. The ratio of the ACTB copies in both replicates ranged from .73 to 1.17 (one outlier of 1.42 removed) for the multiplex assay and from .74 to 1.19 for the singleplex assay. The medians of these ratios were close to 1 for both the singleplex and multiplex assays, i.e. 1.01 and 1 respectively, as could typically be expected for a distribution of intra-run noise. Moreover, the 2 equal aliquots generated similar output in terms of ACTB copy numbers for both the singleplex and multiplex assays separately . Hence, copy numbers from both aliquots were averaged in all subsequent analyses.Thirty cancer positive tissue samples and 12 controls from cancer-free individuals were analyzed with 4 singleplex assays versus 1 multiplex assay. Samples are typically split into 2 equal aliquots to assure output. The control gene ACTB copy numbers were significantly higher for the multiplex assay compared to the singleplex assay , exhibiting a median 1.51-fold copy number increase  and only marginally visible for GSTP1 , which exhibited a median increase of 1.48-fold and 1.08-fold for matched samples, respectively , while the reverse was true for APC . Since the effect of multiplexing on the copy numbers of both RASSF1 and ACTB was fairly similar, the RASSF1 ratios were not altered significantly (p = .96).Q-MSP methylation results are most often reported as a ratio of methylated copies of the target gene divided by the copy number of a control gene, typically GSTP1 copies were observed in all cases, but none of the controls, leading towards a perfect classification and test performance for both the multiplex and singleplex assays , a preferred substitute over test accuracy for small, unbalanced populations [APC versus .78 for the singleplex APC assay, and .42 versus .56 for the multiplex and singleplex RASSF1 assays .A comparison of relative DNA quantity and quality can be made between all these biopsy samples taking into account the tissue volume and the sample age Figure A. The efIn conclusion, the age of samples has a significant impact on the DNA quantity and quality. Increased sample volume is likely to be beneficial to recover more DNA, which could be of noteworthy importance for conducting the assay on older samples. Although larger sample volumes do seem to result in a higher DNA extraction efficiency Figure A, this dACTB copies in either one of the aliquots, of which 10 are classified as older samples (8 from 2005). In addition, 6 of these samples are from 10-micron specimens, 4 from 20-micron biopsies and only 1 from a 40-micron biopsy. Obviously, higher sample quantity and more recent sample fixation, minimize the chance of producing an undetectable ACTB signal. In addition, the majority of the undetectable signals occur in only one of the replicates. When the averaged aliquots are considered instead, only 5 older samples did not contain any ACTB copies, of which three were 20-micron samples and two were 10-micron samples. Figure ACTB copy number. All findings concerning trends for DNA yield relative to sample age and sample quantity are reinforced.With lower amounts of sample input and sample ageing, the likelihood increases that signals will be indistinguishable from noise and therefore lost. Assessing the status of the control gene first, 11 samples failed to show any s 8 from 005. In aACTB copies, that results reliably, i.e. more than 0 copies for both replicates, in the detection of methylated copies of GSTP1, APC and RASSF1 equals to 51, 27 and 63 respectively. A cutoff on ACTB of 50 to 100 copies should result in the reliable detection of methylation of all three genes. Below the cutoff of 63, not detecting methylated copies of either assay could be either due to a true lack of methylation or due to insufficient qualitative DNA. From Figure ACTB copies. For samples up to 5 years of age, a loss can be expected almost regardless of the tissue volume. Allowing a maximal sample loss of 20%, 40-micron samples pose no problem, while 20-micron samples become borderline usable.The minimal DNA quantity, in terms of Probably the most important characteristic of any clinical test is the final sample classification, i.e. whether methylated copies of a test gene can be detected in the case of an epigenetic assay. Key to this is the link between the amount of input material and the possibility of detecting methylation signals. For the tissue minimization study, the methylation ratio was determined using three, matched, biopsy-volume levels, i.e. 10, 20 and 40 microns. If methylation was observed in any of these, the biopsy was considered methylated. Methylation can be missed due to a lack of input material or due to the random sampling technique of the biopsy tissue relative to the tumor’s location within the prostate gland. The random combination of tissue block sections having variable methylation levels could also impact detection.ACTB exceeded 63 copies, as determined above. Table ACTB copies. If sufficient DNA can be detected, the methylation signal was picked up reliably for each assay, down to a volume as low as 20 μm  to determine the limit of detection and analytical sensitivity of the assay. As little as 16 methylated copies of In conclusion, an epigenetic, confirmatory multiplex assay for detecting the presence of prostate cancer cells missed by histology has been further developed. It performed similar relative to the individual singleplex assays for the most important test trait, biomarker gene methylation status. Epigenetic testing can be executed in an accurate and reliable manner in FFPE core tissue biopsies, with as little as 20 μm of sample volume.Thirty tissue samples for the multiplex versus singleplex experiment were obtained during routine prostatectomy as the standard care for 7 patients at the Institut de Pathologie Génétique  and approved by the ethical committees of IPG Gosselies and CHU . Since this is a non-interventional, retrospective, subject-anonymized study, written patient consent was not required by the ethics committees. In addition, 12 cancer-negative BPH (benign prostatic hyperplasia) TURP  samples were obtained from a commercial supplier .Samples for the tissue minimization experiment were obtained from 51 patients, all with Gleason sum 6 (3 + 3) adenocarcinoma of the prostate, undergoing routine prostate biopsy procedures at The Johns Hopkins Medical Center. Testing of these anonymized samples was approved by the IRB at the Johns Hopkins Medical Institutions. Two sets of recent and older specimens were compared, 27 patients’ samples were obtained in 2011, 16 in 2005 and 8 in 2006. For 46 patients multiple, up to 4, cores were available for testing, totaling to 102 unique biopsy samples to be analyzed. Whenever possible, seven 10 micron sections were taken from each of these FFPE blocks, and randomly divided as one tissue biopsy of 40 μm, one of 20 μm and one of 10 μm volume of test tissue.Samples from all cohorts were prepared identically, i.e. cells were scraped from the biopsy slides, followed by deparaffinization  and cell lysis . After the DNA was isolated, it was bisulfite treated , and split into two equivalent replicates for real-time Q-MSP (quantitative methylation specific PCR) analysis .GSTP1APC and RASSF1, either as three separate reactions or multiplexed into one. For each reaction, ACTB was used as a control  and a stock solution containing 800.000 copies per 5 μl was prepared. A serial 10-fold dilution was made down to 8 copies per 5 μl. All 6 standards were included in each PCR run. The vectors contained either the relevant fragment of one gene (singleplex reactions) or all relevant fragments of each one of the 4 genes (multiplex reactions). The standard curves allowed copy number calculations to be performed for each run. Normalized methylation ratios were calculated by dividing the detected amount of methylated copies by the amount of detected All analyses were performed using R and BioConductor, including the ROCR package ,32.BPH, Benign prostatic hyperplasia; FFPE, Formalin-fixed paraffin-embedded; MCC, Matthews correlations coefficient; MSP, Methylation specific PCR; TURP, Transurethral resection of the prostate.This research project was funded by MDxHealth. LVN, JB, GO, JC & WVC are employees of or consulting for MDxHealth and may have corporate stock or stock options. MDxHealth has (pending) patents and exclusive rights on the assay and plans to launch an assay similar to the one described.LVN drafted the manuscript, participated in the design of the study, performed bio-informatics and statistical analyses and interpreted the data. JB participated in the study design, project coordination, helped with data interpretation and to draft the manuscript. AT identified and provided protocol-specific and assisted with project coordination. GO carried out the molecular work, multiplex design and MSP sample testing. JC participated in the study design and project coordination. PD provided samples and participated in the study design. WVC assisted with analyses and data interpretation. JE conceived the study, participated in its design, provided samples and helped to draft the manuscript. All authors read, critically revised and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2490/12/16/prepubVanNesteLetal_Supplementary_BMCUrol_rev1 contains Additional file:Table S1 (primers and beacons) and Additional file :Figure S1 (illustrative ROC curves).Click here for file


Low birthweight should be identified early, even in developing countries where birthweight cannot be easily measured due to the absence of scales and trained staff. This meta-analysis evaluated and compared the use of other anthropometric measurements at birth to predict low birthweight.All studies of medium to high quality  published in English were included. Bivariate random-effects meta-analysis and hierarchical summary receiver operating characteristic curves were used.n = 8, 25, 30, and 6, respectively). Chest circumference and arm circumference had areas under the curve >0.9 (0.95 for both), pooled positive likelihood ratios >5 , and negative likelihood ratios <0.2 ; thigh circumference and foot length were less accurate. There was no substantial difference between chest and arm circumference with respect to pooled sensitivity , specificity , or diagnostic odds ratio . However, as compared with arm circumference, chest circumference showed greater clustering of observations on the hierarchical summary receiver operating characteristic curve and narrower 95% confidence and prediction regions.A total of 69 studies evaluated foot length or the circumference of the chest, (mid-upper) arm, or thigh (Chest circumference and arm circumference have similarly high, although not confirmative, accuracy in predicting low birthweight; however, chest circumference appears to be more precise. However, in some developing countries where home delivery is fairly common despite the high prevalence of low birthweight, it may be difficult to measure birthweight because of inadequate equipment3–13 and a lack of trained health staff.4,5,7,8,13,14 In response to the demand for a rapid, simple, and reliable screening approach for low birthweight, other anthropometric measurements at birth have been studied as surrogates for birthweight, including chest circumference6,8,10,11,14–16 and (mid-upper) arm circumference.3,4,7,9,10,12–19 The number of participants in each of these studies, however, may have been too small to generalize the conclusions to the target populations.7,16 Furthermore, findings vary among studies, and controversy remains regarding the best surrogate measure. A previous meta-analysis found stronger correlations between birthweight and both chest and arm circumferences as compared with other newborn parameters, but did not evaluate the diagnostic performance of these measurements in predicting low birthweight.20 The present meta-analysis utilized pooled sensitivity, specificity, positive and negative likelihood ratios, and the diagnostic odds ratio, as well as hierarchical summary receiver operating characteristic curves, to compare the accuracy of other anthropometric measurements in identifying low birthweight.Low birthweight (<2500 g) is an important public health problem because it is associated with poorer outcomes than normal birthweight.The primary outcomes were the sensitivity and specificity for predicting low birthweight by birth height; head, chest, (mid-upper) arm, abdominal, thigh, and calf circumferences; foot, sternal, sole, and crown-to-rump or crown-to-coccyx lengths; and subscapular and tricipital skinfold thicknesses.21 the PubMed database was searched to locate articles that displayed phrases for the abovementioned anthropometric outcomes in the title or abstract. Each time an article that included 1 or more studies satisfying the selection criteria was identified by scanning the title and abstract, other articles shown under “See all related articles ” on the right side of the web page were also scanned. Articles in the references of already collected articles were also evaluated. MEDLINE, EMBASE, CINAHL, PsychINFO, Wiley InterScience, ProQuest Medical Library, the entire Cochrane Library , and Google Scholar were also used repeatedly . Articles were not excluded due to date of publication. The true positive, false positive, false negative, and true negative values were extracted. When possible, any missing data were calculated by using other existing data, including number of participants, prevalence of low birthweight, and diagnostic indices.The selection criteria were: (1) studies published in English, (2) studies that investigated the diagnostic accuracy of other newborn anthropometric measurements at birth in predicting low birthweight, and (3) studies of high quality . Using the Falck-Ytter filter,22,23 which consists of 14 questions to assess the quality of studies investigating diagnostic performance. The total number of “yes” responses to 14 questions is referred to as the QUADAS score. The Standards for Reporting of Diagnostic Accuracy (STARD) checklist24 was also used to score studies in a similar manner. A study with a QUADAS score of 8 or higher was regarded as eligible, and studies with a QUADAS score greater than or equal to 10 were compared with those with a QUADAS score less than 10 in subgroup analysis. This was done because a QUADAS score of 8 or 10 is commonly regarded as high in meta-analyses published in the most prestigious and other journals,25–28 although neither the QUADAS score, which indicates high quality, nor the numerical methods used to generate scores is uniform.25–28Study quality was assessed using the QUADAS tool,29 The random-effects model allows for heterogeneity among studies. Summary sensitivity and specificity and the corresponding positive and negative likelihood ratios and diagnostic odds ratios were derived from the standard output of the bivariate model, ie, mean logit sensitivity and specificity with their standard errors and 95% confidence intervals (CIs) and the estimates of the between-study variability in logit sensitivity and specificity and the covariance between them. Hierarchical summary receiver operating characteristic curves were simultaneously constructed, and the areas under the curves were also calculated. The ideal cut-off points were derived from the Youden Index, defined as the point on the summary receiver operating characteristic curve that is the farthest from the straight line (representing “area under the curve = 0.500”) that passes through the origin with a 45° angle relative to the Y-axis.30 Heterogeneity was assessed using I2: a value of I2 > 50% was considered to indicate substantial heterogeneity. Sensitivity analysis was conducted to identify sources of heterogeneity in the process of selecting the studies depending on (a) “yes” only or (b) “yes” or “unclear” responses to each of the 14 items on the QUADAS. Publication bias was assessed using Deeks’ funnel plot asymmetry test.31 Subgroup analysis was conducted to assess whether pooled sensitivity or specificity significantly differed by certain study characteristics, namely, Asia vs. other regions, presence vs. absence of a 2 × 2 table, and a QUADAS score greater than or equal to 10 vs. a score less than 10. Stata/SE 11.1 (StataCorp) was used for all analyses.A bivariate random-effects model was used to pool sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio. Logit-transformed sensitivity and specificity  were integrated.3,4,6–19 The study regions comprised Africa, Asia, Europe, and the Middle East, but not Latin or North America. In evaluating birth height; head, chest, arm, thigh, and calf circumferences; and foot length, the total number of participants was 703, 609, 37 293, 22 615, 1964, 624, and 13 120, respectively, while the total number of low-birthweight births was 121 (17%), 105 (17%), 7950 (21%), 3732 (17%), 476 (26%), 126 (20%), and 3752 (29%), respectively. The total number of positive results for the index test was 163 (23%), 219 (36%), 10 458 (28%), 5933 (24%), 453 (23%), 172 (28%), and 5472 (42%), respectively. No study was blinded to the index or reference test(s), but all studies chose the correct reference tests, measured the reference tests independent of the results of the index tests, and interpreted the tests by using the same clinical data as in practice  .32 These estimates, however, did not demonstrate a confirmative level of accuracy .32 Thigh circumference and foot length did not satisfy the criteria for satisfactory diagnostic accuracy due to their lower positive likelihood ratios  and higher negative likelihood ratios . There were too few studies of good quality to evaluate other anthropometric measurements .Both chest and arm circumferences had high sensitivity and specificity Table  and satiI2 ≥ 90%) for both chest and arm circumferences, thigh circumference (98%), and foot length (100%). The pooled estimates were not homogeneous in sensitivity analysis when only “yes” or “unclear”/“yes” responses were used for questions on the QUADAS or when potential outliers were omitted. On the hierarchical summary receiver operating characteristic curves, the observations for chest circumference were more clustered than those for arm circumference  and −4.92 , respectively. The criteria for both high accuracy and strong evidence of diagnostic performance were satisfied in almost all subgroups  vs. other study regions  and a QUADAS greater than or equal to 10  vs. less than 10 .The Deeks’ funnel plot asymmetry test showed that the regression coefficients for assessing publication bias with regard to chest and arm circumferences were 16.4  to investigate articles in the section headed “See all related articles ” on the right side of the PubMed web page when an eligible article was displayed on that web page, (2) to use search engines other than PubMed, and/or (3) to investigate citations in articles that have already been collected, whenever possible. Although blinding to the index or reference test(s), which is a likely cause of bias,33–35 was not done in any of the included studies, the use of the same reference test given for all participants regardless of the results of the index test , the use of clinical populations rather than a diseased population plus a control group, and prospective data collection  were confirmed in almost all the studies .The number of articles increased to 45 after selecting 21 eligible articles via a PubMed search  was smaller than in studies of arm circumference (n = 30). The variations in accuracy in different study groups suggest that extrapolation of overall pooled estimates to individual regions may not always be possible.16 The Deeks’ funnel plot asymmetry test showed absence of publication bias with regard to both chest and arm circumferences, while the inclusion of a sufficient numbers of studies allowed for the statistically significant standard of a P value of less than 0.05, as formally determined (n > 20).36 The test results must be interpreted with caution, however, particularly because the diagnostic odds ratios were very heterogeneous.31,37Based on the present criteria, both chest and arm circumferences had high accuracy and strong evidence of diagnostic performance overall, although they may have lacked confirmative accuracy. Predictive accuracy appeared not to differ between these 2 measurements, as previously reported.e Figure . The gree Figure . This no30 , although it was strong with cut-off points within that range . These findings are additional evidence of the superiority of chest circumference over arm circumference.The frequently used cut-off points for chest and arm circumferences are in accordance with the Youden indicesn = 1) and the insufficient number of studies of arm circumference that explicitly enrolled premature babies (n = 3). Four or more such studies were needed for bivariate diagnostic meta-analyses.Identifying differences in diagnostic performance between studies that did and did not evaluate premature babies is an important goal. This was not possible in the present meta-analysis, however, because of the lack of eligible studies of chest circumference that explicitly excluded premature babies (n = 25 or 30). An extensive literature search was performed by investigating links to related articles on PubMed pages and by using multiple search engines. In addition, true positive, false positive, false negative, and true negative values were extracted whenever possible, even when data were not complete. In addition, large-scale studies were analyzed, including 1 study with as many as 5478 participants.6 The studies encompassed populations in Africa, Asia, Europe, and the Middle East, and the population was thus likely to be racially mixed. Second, bivariate random-effects meta-analysis was used to generate informative estimates. This bivariate model incorporates the correlation between sensitivity and specificity , which is not usually investigated.29 Additionally, 2-dimensional 95% prediction contours, which are also not utilized in conventional analyses, were used in addition to summary operating points , which were calculated from a large number of studies .20This meta-analysis has the following strengths. First, the findings are likely to be generalizable due to the large number of included studies  were almost always controlled for, although a less important cause of bias  was present. Furthermore, meta-regression suggested that studies of higher quality  substantially improved the sensitivity and diagnostic odds ratio of chest circumference (This meta-analysis used data from studies of medium to high quality to evaluate the identification of low birthweight by other anthropometric measurements. It was possible to pool the diagnostic indices for chest, arm, and thigh circumferences, and foot length; however, good-quality studies of other measurements were lacking. In sum, both chest and arm circumferences appear to have high accuracy and strong evidence of diagnostic performance in identifying low birthweight, and there was no substantial difference in accuracy between these two measurements. In contrast, thigh circumference and foot length were less accurate. According to hierarchical summary receiver operating characteristic curves, chest circumference was more precise than arm circumference; therefore, health workers and policy makers may favor this measure over arm circumference.


J. Med. Virol. 84:624–631, 2012. © 2011 Wiley Periodicals, Inc.Acute flaccid paralysis (AFP) surveillance has been conducted as part of the World Health Organization (WHO) strategy on poliomyelitis eradication. Aside from poliovirus, which is the target pathogen, isolation, and identification of non-polio enteroviruses (NPEVs) is also done by neutralization test using pools of antisera which can only identify limited number of NPEVs. In the Philippines, despite the significant number of isolated NPEVs, no information is available with regard to its occurrence, diversity, and pattern of circulation. In this study, a total of 790 NPEVs isolated from stool samples submitted to the National Reference Laboratory from 1992 to 2008 were analyzed; neutralization test was able to type 55% (442) of the isolates. Of the remaining 356 isolates, which were untyped by using neutralization test, 348 isolates were analyzed further by RT-PCR targeting the VP1 gene. A total of 47 serotypes of NPEV strains were identified using neutralization test and molecular typing, including 28 serotypes of human enterovirus B (HEV-B), 12 serotypes of HEV-A, and 7 of HEV-C. The HEV-B group  constituted the largest proportion of isolates, followed by HEV-C , HEV-A , and no HEV-D. Coxsackievirus (CV) B, echovirus (E)6, E11, and E13 were the most frequent isolates. E6, E11, E13, E14, E25, E30, E33, CVA20, and CVA24 were considered as endemic strains, some NPEVs recurred and few serotypes existed only for 1–3 years during the study period. Despite some limitations in this study, plural NPEVs with multiple patterns of circulation in the Philippines for 17 years were identified.  HEVs are small, non-enveloped RNA virus that is the largest member of the family Picornaviridae. More than 90 antigenically distinct serotypes are known to cause infections in humans and based on the pathogenicity in humans and in experimental animals, enteroviruses were classified previously as polioviruses (PVs), coxsackieviruses A and B (CVA and CVB), echoviruses (E), and the numbered enteroviruses (EV). On the basis of nucleotide and amino acid sequences of the viral protein genes, a new classification was proposed and HEVs have been subgrouped into seven species namely HEV-A (17 serotypes), human enterovirus B (HEV-B) (58 serotypes), HEV-C (16 serotypes and three PVs), and HEV-D (three serotypes) and rhinovirus A, B, and C  to be less important as human pathogens worthy of sustained investigation compared to other viral infections or diseases of greater perceived public health importance. However, NPEVs can cause a broad spectrum of clinical conditions and may also result in serious or even fatal outcome. Enteroviruses are viruses implicated most commonly in of acute myocarditis, acute hemorrhagic conjunctivitis, hand–foot–mouth disease with neurological complications and aseptic meningitis [Goh et al., One of the key strategies for eradication of poliomyelitis set by World Health Organization (WHO) is laboratory-based detection of PV through acute flaccid paralysis (AFP) surveillance. In line with this WHO strategy, the national AFP surveillance program was established in the Philippines in 1992. Since then, this surveillance system has been the only source of information on EV infection for the country. Laboratory test for EV is conducted for stool specimens from patients with AFP. The last indigenous wild-type polio was detected in 1993 and the Philippines was declared polio-free in 2000. Through the years, poliomyelitis cases have been reduced worldwide and eradicated in some parts of the world, leaving the NPEV as one of the implicated causes of AFP. In the Philippines, NPEVs account for about three-fourths  of the total isolates from 1992 to 2008 in AFP surveillance. Among these NPEVs, almost half were untypable using WHO protocols on enterovirus detection . Identifying the serotype circulating in the country may help in understanding epidemiology of enterovirus infection and to a larger context of polio eradication, may give the assurance that PV is not overlooked. Laboratory data were analyzed to give an overview of identified NPEV serotypes and their occurrence, diversity, and pattern of circulation by showing the results during a 17-year study period as implicated in AFP in the Philippines.A total of 11,079 stool samples collected from 5, 268 children aged <15 years who were presenting acute flaccid type of paralysis from January 1992 to December 2008 were sent to the WHO National Reference Laboratory for PVs at the Research Institute for Tropical Medicine. Most of the cases had two stool specimens with an interval of 24–48 hr between collections and within 14 days from date of onset of paralysis. Out of these stool samples, 798 NPEVs (7.2%) were isolated during the study period of 17 years, however, only 790 isolates were included for analysis in this study since other eight samples could not be found.Initially, the neutralization test was carried out on the 798 NPEVs by using the pools of antisera. Serotyping was done on all RD-positive isolates according to the time frame set by WHO that result must be released within 28 days of receipt of the specimens. The method enabled serotype identification of 55% (442) of the NPEV isolates. From review of records, the remaining isolates including the missing eight samples (356) yielded no neutralization pattern and were thus reported as untypable NPEV (uNPEV). Since the present study is not part of the routine surveillance program on the basis of typing all NPEVs, a microneutralization test was not repeated, instead, the remaining 348 uNPEV isolates were analyzed by a molecular method.All samples were inoculated onto the WHO-recommended cell lines for HEV detection: RD and HEp2C (Cincinnati) cell lines were used from 1992 to 1997 and from 1997 onwards, RD and L20B cells were used. L20B, a genetically engineered mouse cell line, is intended for the specific isolation of PV but recently, it has been reported that it can also propagate some CVA types 4, 8, and 10 [Nadkarni and Deshpande, Microneutralization tests with pools of antisera specific for common EV serotypes were used for all the virus isolates obtained, according to WHO standard protocols. Serotype-specific immune antisera for the CVB and some echovirus serotypes which were available only until 2000 and EV pools from the National Institute for Public Health and the Environment, Bilthoven, the Netherlands were used. The EV pools contain reference horse typing antisera against the HEV serotypes isolated most frequently combined as nine antiserum pools: polyclonal antisera against a trivalent pooled polio antiserum (PP pool), a CVB1-6 pool (CP pool), a CVA9, and echoviruses (A-G pool). The pools can only identify 27 out of more than 90 existing HEV serotypes. Briefly, diluted isolates were mixed with equal volumes of the aforementioned antisera pools. Daily microscopic observation was performed to check for the presence of characteristic rounding necrosis CPE. The antiserum that prevented the development of CPE indicated the identity of the virus WHO, . The iso2O. Thermocycling was performed in GeneAmp® PCR System 9700  for 50°C for 30 min, 94°C for 3 min followed by 30 cycles at 94°C for 30 sec, 42°C for 30 sec, and 72°C for 30 sec with ramp time of 1 min. The reaction products were analyzed by electrophoresis in a 2% Agarose/0.5 TBE gel stained with ethidium bromide. All VP1 positive samples with the expected amplicon size were subjected to purification using SUPREC PCR (Takara). Purified products were then labeled directly by cycle sequencing reaction with BIG DYE Terminator v.1.1 and were analyzed by ABI 3730 Genetic Analyzer (Applied Biosystems).Reverse transcriptase-polymerase chain reaction (RT-PCR) on 5′ UTR and partial sequencing of the VP1 capsid region were performed for the 348 isolates which were isolated on RD cells and previously reported as uNPEV. In brief, viral RNA was extracted from culture supernatants using Purelink Viral DNA/RNA kit  according to the manufacturer's instruction. Thereafter, complementary DNA was synthesized using extracted RNA with random primers, M-MLV, and RNAse OUT (Invitrogen). The 5′ UTR was amplified and used as screening test for enterovirus and exclusion as described previously [Iturriza-Gómara et al., Automated DNA sequencing of the amplified fragments was carried out on both strands and alignment of the obtained sequences with selected reference serotypes from GenBank was performed using the Clustal W algorithm as implemented in MEGA version 4.0 [Tamura et al., Due to the variation in the total number of specimens received and isolates detected annually, a priori cut-off point was set to define patterns of circulation during the study period: endemic is defined when a serotype was isolated for at least 8 years; cyclic are those serotypes which were isolated for at least 4 years; epochal are those serotypes which occurred in 1–3 years, within the 17-year period.Since the nationwide surveillance for AFP was implemented in the Philippines in 1992 up to 2008, 798 NPEVs (7.2%) were isolated during the study period of 17 years. Microneutralization test enabled the serotype identification of 55% (442) of the NPEV isolates, mostly belonging to HEV-B group. The remaining isolates (356) yielded no neutralization pattern and were thus reported as uNPEV.The viral genomes of 348 samples were positive for the 5′UTR and VP1 regions. The sequences obtained in VP1 were compared with those included in GenBank database and were assigned the serotype of the strain that gave the highest identity score. The sequences revealed their serotypes, when homology in VP1 sequence was at least 75% to prototype strains.Results of serological and molecular tests were summarized in During the study period, the NPEV mean isolation rate was 7.8%, with only 4 years  reaching more than 10% . Of the E11was the most predominant serotype followed by, in descending order, E13, E7, E6, CVA24, E30, E19, E29, CVA20, and E7, all of which were identified almost annually except E19, E29, E7, and E3, which appeared to be recurrent. Other serotypes  have the tendency of recurrence at variable time intervals, ranging from 4 to 7 years. Also, epochal viruses, most of which were identified just recently  and rare serotypes (CVA8 and CVA14), have been observed during the study period. Notably, using the criteria for circulation pattern, no HEV-A was identified as endemic. HEV-B circulated predominantly as endemic, cyclic, and epochal; similarly, HEV-C in the Philippines showed three defined circulation patterns but with few isolates.WHO protocols for enterovirus detection and identification for AFP surveillance are based on virus isolation followed by serotyping the isolated viruses by neutralization test which allows the identification of only 27 enterovirus serotypes [Muir et al., The incidence of different species of enteroviruses in different countries is shown in The results indicated that multiple patterns of circulation of NPEV in the Philippines over 17 years existed and that, plural serotypes co-circulated each year. Circulation of individual serotypes varied from endemic, cyclic, and epochal patterns . The cycIn this study, recently identified EVs have been documented: EV73, EV74, EV76, EV77, EV80, EV90, EV96, and EV99. These viruses along with E19, CVA24, and E24 are not included in the EV antisera pools. It can be noticed that activities of E29 (2001), CVA24 (2000), E7 (2000), and E3 (1997) were marked with high isolations. However, review of records showed that E29 isolations were collected on almost the same week with only 2 days interval (data not shown) in Region 10 of the Philippines. Though the data might suggest that there was a circulation of E29 in a community in 2001, a possible outbreak cannot be dismissed. In addition, HEV-A and HEV-C and PV 1 isolates were missed by serologic test and were identified through molecular method. The polio isolate was confirmed later as Sabin-like by both the regional  and global  reference laboratories. Record-tracing revealed that the PV detected which was isolated and reported as uNPEV in year 2000 had a negative result on the first inoculation in both RD and L20B cell lines and on re-passage, showing characteristic CPE only in RD but not in L20B. The event prompted the laboratory to proceed on performing the microneutralization test which resulted into no neutralization pattern in EV pools . After the molecular identification of PV 1 in the present study and before sending to the global reference laboratories for further characterization, verification of the true identity of the PV was done by reinoculating the samples onto RD as well as L20B and by serotyping using monospecific polyclonal PV antisera to types 1, 2, and 3 combined as antiserum pools. Both cell lines, RD and L20B, presented the characteristic CPE and microneutralization test concluded that the sample was indeed, a PV 1. While the polio case was missed, the event points out two aspects: (i) it is a vital component that proper inoculation of samples must be ensured and that vigilance while performing the experiment, i.e., inoculation, must be observed; (ii) it underlines the importance of employing molecular methods to help us interpret trends and patterns and elucidates the partial, if not actual, burden of disease caused by NPEVs and their contribution to the PV surveillance and as the goal of eradication is approaching, to assure that PV cannot be missed.The isolation of rarely reported enteroviruses like CVA3, CVA8, CVA13, CVA14, CVA17, CVA19, CVA20, CVA21, E12, E29, EV73, EV74, EV76, EV77, EV80, EV90, EV96, and EV99 during the study period was observed. According to epidemiological data on EVs from Europe, USA, Latin America, China, and Japan, these viruses have limited isolation frequency, with only <100 detections worldwide [Infectious Disease Surveillance Center, NPEVs are common in children, however, its importance in the Philippines has not been well documented even if large outbreaks occurred in its neighboring countries. For example, E11, E13, and E30 are well known among the serotypes of EVs causing aseptic meningitis and along with EV71 which is the causative agent of hand–foot–mouth disease with some severe complications. These outbreaks due to NPEVS were commonly reported in the Western-Pacific countries like Japan, Korea, Taiwan, Malaysia, and China Kit, . Some ofThe minimum NPEV rate or sensitivity measurement for PV surveillance set by the Western Pacific Region is greater than 10% WHO, . The setThis study is subject to a number of limitations. First, detection of EV in line with the AFP surveillance is the only data source where occurrence of EV infections could be traced back. Enterovirus surveillance encompassing all related infections has not taken place in the Philippines. Thus the data may be underrepresented of the overall impact of NPEV infection in the country. Furthermore, overrepresentation of neurotropic NPEV types may probably bias the data if AFP cases will be the only core of identifying NPEVs. Nonetheless, their detection may either reflect their causative role in AFP or the possibility that these enteroviruses just transiently localize the gastrointestinal tract and are being shed in stool specimen. Second, some CVA strains that cannot be propagated in RD and HEp2C may have been under-detected and the growth of other EV types may have been favored [Hosoya et al., Based on the nationwide surveillance of AFP-associated infections in the Philippines, this study examined systematically the datasets since 1992–2008 in order to elucidate the proportion, diversity, circulation pattern of NPEVs. In conclusion, although EV detection is only a “part-outcome” of the PV surveillance, different patterns of circulation of plural enterovirus serotypes were detected.