Previous studies have reported seasonal variation in peptic ulcer disease (PUD), but few large-scale, population-based studies have been conducted.per se in the region. For statistical analysis, the χ2 test for goodness of fit and inferential chronobiologic method  were used.To verify whether a seasonal variation in cases of PUD (either compicated or not complicated) requiring acute hospitalization exists, we assessed the database of hospital admissions of the region Emilia Romagna (RER), Italy, obtained from the Center for Health Statistics, between January 1998 and December 2005. Admissions were categorized by sex, age , site of PUD lesion (stomach or duodenum), main complication (hemorrhage or perforation), and final outcome . Temporal patterns in PUD admissions were assessed in two ways, considering a) total counts per single month and season, and b) prevalence proportion, such as the monthly prevalence of PUD admissions divided by the monthly prevalence of total hospital admissions, to assess if the temporal patterns in the raw data might be the consequence of seasonal and annual variations in hospital admissions p < 0.001)], 7,151 were <65 yrs of age, 8,849 between 65 and 74 yrs of age, and 10,848 ≥ 75 yrs of age. There were more cases of duodenal (DU). (89.8%) than gastric ulcer (GU) (3.6%), and there were 1,290 (4.8%) fatal events. Data by season showed a statistically difference with the lowest proportion of PUD hospital admissions in summer (23.3%) (p < 0.001), for total cases and rather all subgroups. Chronobiological analysis identified three major peaks of PUD hospitalizations  for the whole sample (p = 0.035), and several subgroups, with nadir in July. Finally, analysis of the monthly prevalence proportions yielded a significant (p = 0.025) biphasic pattern with a main peak in August-September-October, and a secondary one in January-February.Of the total sample of PUD patients  is observed. When data corrected by monthly admission proportions are analyzed, late summer-autumn and winter are confirmed as higher risk periods. The underlying pathophysiologic mechanisms are unknown, and need further studies. In subjects at higher risk, certain periods of the year could deserve an appropriate pharmacological protection to reduce the risk of PUD hospitalization. Peptic ulcer disease (PUD) is representative of a group of ulcerative disorders of the upper gastrointestinal tract (GIT), mainly involving the stomach and duodenum, that share a common acid-pepsin pathogenesis . The maj2, has a total population of ~3,985,000 people , with a density of 180 persons/Km2.The study was conducted with the approval of the local institutional committees for human research. The analysis included all consecutive hospital admissions for PUD that occurred between January 1, 1998 and December 31, 2005, as recorded in the database of the RER Center for Health Statistics. The RER is situated in north-eastern Italy and has a surface area of 22,124 KmInternational Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM). The RER health authorities removed patient name, exact address, and other potential identifiers from the database provided for this study, to respect national dispositions-by-law in terms of privacy. Birth date was the only identification data allowed for analysis, in order to categorize the admissions by age group and to clean the database of potential repeat hospital admissions of the same person. We sought to include in the statistical analyses only the calendar date of the first hospital admission of any given PUD crisis per patient, to adhere to the statistical assumption of independency of observations. It is possible, however, that data of different (multiple) patients with a same birth date could have been inappropriately deleted because they were believed to be a readmission for an ongoing PUD crisis of the same person, but the occurrence of such cases was rare, with no potential impact on the findings, given the almost 27,000 total PUD admissions considered. Of course, since the electronic RER database started on 1998, no information is available on cases of PUD admissions prior to that year.Starting from 1998, the RER elaborated an electronic database tracking all discharge hospital sheets  of persons admitted to public and private hospitals, which collected an overall total of ~5.6 million cases as of the latest updated database (December 2005) used for this investigation. The DHS lists the name, sex, date of birth, date of hospital admission and discharge, department of admission and discharge, up to nine discharge diagnoses, and the most important diagnostic procedures coded according to the The unit for the statistical analysis of the data was admission date for acute PUD as per ICD-9-CM codes 531.00 to 534.91. Only admissions directly related to PUD, i.e., only cases in which PUD was indicated as the main discharge diagnosis, were abstracted from the database. The total sample of hospital admission data was divided into subgroups categorized by sex, age , site of lesion -- gastric ulcer or GU (ICD-9-CM codes from 531.00 to 531.91), duodenal ulcer or DU (ICD-9-CM codes from 532.00 to 532.91), peptic ulcer (PU), site unspecified (ICD-9-CM codes from 533.00 to 533.91), and gastrojejunal ulcer (ICD-9-CM codes from 534.00 to 534.91). They were also categorized by main complication, i.e., hemorrhage , perforation , and a rough indicator of outcome, i.e., fatal: in-hospital death, and nonfatal: patient discharged alive.We used SPSS 13.0 for Windows, SPSS Inc., Chicago, IL, 2004 for statistical analysis of demographic data, e.g., age comparisons between men and women.per se in the region.Temporal patterns in PUD admissions were assessed in two ways, considering a) total counts per single month and season , and b) prevalence proportion, such as the monthly prevalence of PUD admissions divided by the monthly prevalence of total hospital admissions, to assess if the temporal patterns in the raw data might be the consequence of seasonal and annual variations in hospital admissions midline estimated statistic of rhythm , amplitude , and peak (acrophase) and trough (bathyphase) time referenced to 00:00 h December 31. Significance levels were set at p < 0.05.The distribution of the total and subgroup PUD hospital admissions within the four 3-month periods (seasons) of the year was tested for uniformity by the χ2 test for goodness of fit. Analysis of annual variation in admissions was performed by applying a partial Fourier series to the time series data using an internationally validated method (Chronolab software on an Apple Macintosh computer) . This mep < 0.001). PUD admissions were more commonly associated with advanced age; 7,151 were <65 yrs of age, 8,849 between 65 and 74 yrs of age, and 10,848 >75 yrs of age. There were more cases of duodenal (DU). (89.8%) than gastric ulcer (GU) (3.6%). As for outcome, there were 1,290 (4.8%) admissions culminated in an in-hospital death. Overall, 15,223 cases of complications were associated with complications, 12,682 being hemorrhage (47.2%) and 2,541 (9.5%) perforation.Between January 1, 1998 and December 31, 2005, the database of the RER identified 26,848 hospital admissions corresponding to ICD-9-CM codes indicative of PUD; 16,795 (62.5%) were males (mean ± SD) 65 ± 16 yrs of age and 10,053 (37.5%) females 72 ± 15 yrs of age . The findings of this objective inferential time series analysis are representative of the raw data shown in Figure p = 0.032), 65-74 yrs of age (p = 0.022), GU (p = 0.002), and DU (p = 0.036). With the exception of a few subgroups, the nadir of admissions consistently occurred in the month of July  for the entire sample of admissions p = 0.05. The fip = 0.095). The seasonal variation in total hospital admissions, i.e., between the spring peak and summer trough, amounts to ~13%. Figure p < 0.0001). Inferential analysis of the PUD hospital admission rate data, exploring the fundamental 1 yr and up to three best fitting harmonics, reveals a statistically significant (p = 0.025), modest-amplitude  annual variation with the major peak in August-September and secondary peak in January-February. The findings of the objective inferential analysis are representative of the monthly rate data shown in Figure The PUD data were also analyzed in terms of monthly prevalence proportion, i.e., the monthly prevalence of PUD admissions divided by the monthly prevalence of total admissions. Figure Helicobacter pylori changed the perspective that PUD is simply a disorder of gastric acid homeostasis. Considerable research has clearly established that H. pylori is a significant cause of gastritis and PUD. Indeed, acid hypersecretion, which is commonly observed in PUD, is more likely the result of H. pylori infection rather than being the primary abnormality [Peptic ulcerations of the gastro-duodenal tract are defects of the mucosal lining resulting from the epithelial cell damaged evoked by acid and pepsin as caustic agents . From thormality . Recent ormality . Peptic ormality ,15. Baseormality ,17. Seveormality -22 also Although seasonal variation in PUD onset has been rather extensively investigated, the conclusions of the previous studies, with the exclusion of two large investigations performed in the USA  and in CSeasonal variation in PUD, either complicated or uncomplicated, and typically investigated as the number of cases  per month or season, has been a common research topic over the years, Table . AutumnaWhy PUD is more frequent in winter and late summer-autumn months? Although the answer is far from being conclusive several factors have been claimed to play a role in PUD seasonality. Concerning severe PUD, Nomura et al.  found thH. pylori infection is another possible factor which may affect PUD onset. Savarino et al. [H. pylori-positive DU cases between seasons or a parallel annual fluctuation in gastric acidity and H. pylori infection. These findings are in line with another study showing no significant correlation between the seasonal differences in the diagnosis of ulcer disease and presence of H. pylori infection [Seasonality of o et al. , howevernfection . Whethernfection  found thnfection  for ketonfection  showing nfection  found thAlcohol is another powerful gastrolesive variable in ulcerogenesis, and it is plausible that seasonal differences in alcohol consumption might contribute to seasonality of severe PUD hospital admissions. However, the seasonal pattern in drinking behavior has not been extensively studied. The only indirect available evidence reported a winter to summer increase in per-capita alcohol sales in the U.S.A. , and a hSome previous studies reported a seasonal pattern for acute PUD, mainly characterized by autumn and winter peaks. The present large-scale, population-based study provides evidence of an annual pattern of PUD hospitalizations in the RER, characterized by peaks in autumn, spring and winter, when raw data were considered. However, when the analysis was done on the prevalence proportions, such as the monthly prevalence of PUD admissions divided by the monthly prevalence of total hospital admissions, a biphasic pattern was shown, with a main peak in late summer-autumn (August-September-October) and a second one in winter (January-February).H. pylori infection may have an impact in different seasonal peaks of PUD worsening. Moreover, very little is known about the possible influence of biological rhythms on the occurrence of acute PUD. Although this study does not provide insights into pathogenetic factors, the possible demonstration of seasonal variation in PUD hospital admissions may prompt further research to better understand seasonality and guide appropriate management.We are aware of the limitations of the present work, including lack of data on: 1) weather variables of the RER (characterized by a large extension and wide orographic and climatic conditions); 2) regional  seasonal variation in the severity of patient co-morbidities ; 3) alcohol consumption. It is possible, in fact, that susceptibility to different factors, such as NSAIDs, alcohol consumption and The authors declare that they have no competing interests.RM, RDeG, MHS, RC, MG: conceived of the study, participated in the design and coordination of the study, performed the statistical analysis, and participated to draft and critically revise the manuscript. BB, RS, MS, GB, VS: participated in the design of the study, obtained data, participated in the statistical analysis, and participated to draft the manuscript. DF, EC: participated in the design and coordination of the study, helped for obtaining data, and participated to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/10/37/prepub


ADD1) gene have been implicated as a risk factor for hypertension. Given its clinical significance, we investigated the association between ADD1 Gly460Trp gene polymorphism and essential hypertension in an Indian population. Further, a meta-analysis was carried out to estimate the risk of hypertension.Essential hypertension is a complex genetic trait. Genetic variant of alpha adducin (ADD1 gene polymorphism. Genotyping was determined by real time PCR using Taqman assay. Multiple logistic regression analysis was used to detect the association between Gly460Trp polymorphism and hypertension.In the current study, 432 hypertensive cases and 461 healthy controls were genotyped for the Gly460Trp No significant association was found in the genotype and allele distribution of Gly460Trp polymorphism with hypertension in our study. A total of 15 case-control studies were included in the meta-analysis. There was no evidence of the association of Gly460Trp polymorphism with hypertension in general or in any of the sub group.ADD1 polymorphism may not be excluded by a negative association study. Further, large and rigorous case-control studies that investigate gene–gene–environment interactions may generate more conclusive claims about the molecular genetics of hypertension.We found that the Gly460Trp polymorphism is not a risk factor for essential hypertension in a south Indian Tamilian population. However, the role of  Essential hypertension is a highly prevalent, complex, multifactorial disorder that arises from the interaction of genetic predisposition and environmental risk factors. It is significantly associated with the pathophysiology of cardiovascular disorders such as myocardial infarction, stroke, and coronary artery disease. Epidemiologic studies revealed that 20–40% variation in blood pressure is due to genetic heritability.2ADD1) gene. Adducin, a cytoskeleton component, is a heterodimeric protein present in many tissues with α, β and γ subunits involved in cell to cell contact, cell membrane ion transport and signal transduction.[ADD1 is one among the proteins that regulate Na+-K+ ATPase activity. Abnormalities in adducin by genetic mutation have been shown to influence the surface expression and maximum velocity of Na+-K+ ATPase and subsequently faster renal tubular Na reabsorption.[ADD1 in the pathogenesis of essential hypertension both in human and animals.[Numerous genetic markers have been identified in the regulation of blood pressure and essential hypertension. One suchsduction.–6 ADD1 isorption. Clinical animals.ADD1 is mapped onto the chromosome location 4p16.3. The common molecular variant of the ADD1 gene causing the substitution of tryptophan instead of glycine (Gly460Trp) at amino acid position 460 was found to be associated with increased risk of hypertension[ADD1 gene polymorphism and susceptibility to essential hypertension but the results have been inconsistent and inconclusive. Some studies implicated the positive association of the Gly460Trp polymorphism,[The gene encoding human ertension and otheertension and leftertension There armorphism,–15 wheremorphism,ADD1 gene polymorphism in hypertension worldwide and there is paucity of data relevant to the Indian population. In view of the above, we carried out a case-control study to investigate the association of ADD1 gene Gly460Trp polymorphism and susceptibility to essential hypertension in a south Indian Tamil population. In addition, a meta-analysis was conducted to examine the prevalence of Gly460Trp polymorphism comprising 45 studies.Several studies have described the role of The study was carried out in 432 unrelated essential hypertensive cases (212 men and 220 women) aged 30-60 years. They were diagnosed and selected from the outpatient clinics of hypertension and internal medicine . All of them were residents of Tamil Nadu and Pondicherry for at least three generations. Patients receiving antihypertensive medications for more than three months (or) newly diagnosed hypertensive patients with systolic blood pressure more than 140mmHg and/or diastolic blood pressure more than 90mmHg  on two or more consecutive visits were considered to be hypertensives. The age 2 ). Blood pressure was measured by resting the subjects for 10 min in the right arm by using standard sphygmomanometer and the average of three readings taken 2 min apart was recorded. The study was approved by institutional ethics committee and written informed consent was obtained from all the participants.The control group consisted of 461 healthy volunteers (210 men and 251 women) aged 30-60 years. They had no personal or family history of hypertension in the first degree relatives with systolic blood pressure less than 130 mmHg and diastolic blood pressure less than 85 mmHg. Patients who visited the outpatient clinics with minor illness without hypertension, diabetes mellitus, hyperlipidemia and family history of hypertension in previous records were recruited as controls. None in the control group were receiving antihypertensive therapy, treatment for heart disease, or hormone replacement therapy during the time of investigation. Plasma lipid profile and blood glucose level were measured after overnight fasting for both hypertensives and normotensives to rule out diabetes and hyperlipidemia. A detailed family history relating to their pedigree was taken to identify if any of the close relatives of the volunteers or patients were hypertensives. Details such as identification characteristics, body weight and height, drug history were recorded. All the participants were interviewed using a standardized questionnaire with regard to their lifestyle, smoking, alcohol consumption and drug intake. In all subjects, height was measured to the nearest centimeter and weight to the nearest 0.1kg, which was used for calculation of BMI (kg/mFive milliliter of venous blood was collected using ethylene diamine tetra acetic acid (EDTA) as anticoagulant. Genomic DNA was extracted from the peripheral leucocytes using the standard phenol: chloroform method and stored at 4°C. Genotyping for Gly460Trp was carried out by Real Time Thermocycler  using Taqman SNP genotyping assay method. This technique employs fluorogenic 5’nuclease chemistry  to enable detection of specific PCR product. C__11764545_20 was used as the SNP genotyping assay ID . The PCR reaction was carried out in duplicate using 20 µL final volume that contained 10 µL of Taqman Universal PCR master mix (2X), 0.75 µl of 20X working stock of SNP genotyping assay, and 4.5 µL of genomic DNA diluted in DNase free water and 4.75 µL of deionized water. The thermocycler conditions included 1 cycle at 50°C for 2 min; 1 cycle at 95°C for 10 min to activate the AmpliTaq Gold polymerase followed by 40 cycles of denaturation at 92°C for 15 s and annealing/extension at 60°C for 1 min. The allelic discrimination analysis was performed using 7300 SDS software version 1.3.1.P value <0.05 was used as the level of significance.Statistical analysis was done using Statistical Package for Social Sciences software . The demographic details of cases and controls with continuous variables were compared using Student’s unpaired ‘t’ test while dichotomous variables were analyzed using Fisher’s exact and Chi square test. Differences in allele frequencies and genotype distribution between hypertensive and normotensive groups were compared by Chi square/Fishers exact test. The association between genotypes and hypertension risk was analyzed by calculating the crude odds ratio (OR) and 95% confidence interval (95% CI) using Chi square/Fishers exact test. The adjusted odds ratio was calculated using unconditional logistic regression and the low risk genotype was designated as the reference category. The observed genotype frequencies were compared with the expected frequencies to check for the Hardy–Weinberg equilibrium and P<0.001). No significant difference was observed in the sex distribution, BMI, total cholesterol, triglycerides, LDL and HDL cholesterol levels among the cases and controls. However, there were significant differences in age, systolic blood pressure (SBP), diastolic blood pressure (DBP), smoking pattern, alcohol consumption and VLDL cholesterol levels among the cases and controls. Potential confounding variables that revealed significant differences between cases and controls  were taken for logistic regression analysis.The baseline characteristics of the study subjects are shown in P>0.05).The genotype and allele frequencies are shown in vs. 46.8 ± 0.5, P<0.02). Smokers  and alcoholics  were higher in male cases as compared to male controls. These confounding variables were adjusted by logistic regression analysis to calculate the adjusted odds ratio as shown in Gender specific analysis was carried out by comparing hypertensive men and women with their respective control groups as shown in ADD1 gene comprising 45 studies[1122A meta-analysis was conducted on Gly460Trp polymorphism of the studies11–2224–55 ADD1 Gly460Trp polymorphism and essential hypertension by combining 15 case-control studies, involving 3187 cases and 3720 controls[9229Further, we also analyzed the association between controls9–2224–27 trols922914 In theCardiovascular disease is the leading cause of death in both the economically developed and developing countries. The recent epidemiological investigation on Indian population has shown that the incidence of cardiac events and cardiac mortality would be doubled by the year 2020. In India, hypertension directly accounts for about 57% of stroke and 27% of CAD deaths.ADD1 gene and susceptibility to essential hypertension in an Indian population. Our data showed that the genotype and allele frequencies were similar between hypertensive cases and controls and were not associated with hypertension.To the best of our knowledge, the present study is the first and largest case-control study to address the role of Gly460Trp polymorphism of the Despite the difference in the variant allele frequency, the results of the present study were in accordance with other studies conducted in Japanese, Chinese, Koreans and Caucasians.18202124 1820219ADD1 Gly460Trp gene polymorphism and essential hypertension was first reported by Cusi et al, in white population. They observed that the Gly460Trp polymorphism is associated with salt sensitive form of hypertension.[ADD1 Gly460Trp polymorphism in the etiology of essential hypertension. However, varied results have been obtained.[Significant association between rtension. Later onobtained.–22ADD1 may affect blood pressure by modulating renal tubular reabsorption of sodium through the activation of Na+, K+, -ATPase with the ADD1 460Trp exhibiting higher affinity for the Na+, K+, -ATPase pump. Several studies reported the relationship between ADD1 polymorphism and anti-hypertensive drug response.[445744ADD1 W allele + AGT T allele + diuretics) significantly reduced DBP in hypertensive cases after diuretic therapy.[It has been postulated that response.445758 Sponse.44574457 On tADD1 polymorphism predicted the high risk of mortality in type 2 diabetic patients and increased mean common carotid IMT in relation to hypertension.[In the Rotterdam study, the rtension. Furthermrtension. Similarlrtension. In contrrtension. A large rtension. In additrtension. Factors ADD1 polymorphism, meta-analysis is not available so far. In the present meta-analysis, the occurrence of variant allele 460Trp was higher in Asians and lower in Africans, a trait that did not match with other ethnic groups. In addition, the consistency of genetic effects across populations from different ethnicities was also investigated. Race was a major determinant of the Gly460 and 460Trp allele frequencies. The overall lack of association between Gly460Trp ADD1 gene polymorphism and hypertension and the discrepancy of results between Caucasians, Asians and blacks might be because of other unidentified polymorphisms that exist in the ADD1 gene that affect the susceptibility to hypertension or other loci that might be in linkage disequilibrium with the examined polymorphism of ADD1 gene. Rather than the individual mutation, interaction of multiple polymorphisms or haplotypes can be a major determinant of disease susceptibility. Finally, the limitation of the meta-analysis is that our results were extracted directly from published articles and not from the original data provided by the authors.Even though several studies were done on ADD1 variant 460Trp in the causation of essential hypertension suggesting that it is unlikely to be a predisposing factor in hypertension.The strength of our study includes strict selection of cases and controls from homogenous population. In conclusion, neither the present study nor the meta-analysis offers evidence for the


The aim of this study was to compare results of QFT and tuberculin skin test (TST) among newly arrived asylum seekers in Norway and to assess the role of QFT in routine diagnostic screening for latent tuberculosis infection.QuantiFERONThe 1000 asylum seekers (age ≥ 18 years) enrolled in the study were voluntarily recruited from 2813 consecutive asylum seekers arriving at the national reception centre from September 2005 to June 2006. Participation included a QFT test and a questionnaire in addition to the mandatory TST and chest X-ray.Among 912 asylum seekers with valid test results, 29% (264) had a positive QFT test whereas 50% (460) tested positive with TST (indurations ≥ 6 mm), indicating a high proportion of latent infection within this group. Among the TST positive participants 50% were QFT negative, whereas 7% of the TST negative participants were QFT positive. There was a significant association between increase in size of TST result and the likelihood of being QFT positive. Agreement between the tests was 71–79% depending on the chosen TST cut-off and it was higher for non-vaccinated individuals.By using QFT in routine screening, further follow-up could be avoided in 43% of the asylum seekers who would have been referred if based only on a positive TST (≥ 6 mm). The proportion of individuals referred will be the same whether QFT replaces TST or is used as a supplement to confirm a positive TST, but the number tested will vary greatly. All three screening approaches would identify the same proportion (88–89%) of asylum seekers with a positive QFT and/or a TST ≥ 15 mm, but different groups will be missed. M. tuberculosis specific antigens ESAT-6 and CFP10 have recently been developed. These protein antigens are present in all species of the M. tuberculosis-complex (including M. bovis), but absent in all vaccine strains of M. bovis-BCG  and most non-tuberculosis mycobacteria, except M. marinum, M. zulgai, and M. kansasii. These tests can therefore diagnose infection with M. tuberculosis with a higher specificity [®TB Gold (QFT), offers logistic advantages and may be suitable for routine screening. The assay has been tested in numerous contact investigations, among patients with tuberculosis disease and exposed health care workers, but data are limited for immigrant screening [The incidence of tuberculosis in Norway is generally low (6.3/100 000 population in 2006), but high among immigrants from countries where tuberculosis is endemic . The enocificity . One of creening -12. Amoncreening . Vast heAsylum seekers undergo mandatory screening for tuberculosis upon arrival in Norway, with TST and for persons over 15 years also chest X-ray . The covTST was performed according to the Mantoux method with Purified Protein Derivate (PPD): RT 23, 2 TU from SSI, Copenhagen, Denmark. Four experienced nurses applied and read the test. A test was considered positive if the induration was ≥ 6 mm after 72 hours. Reading was double if the indurations were large, showed adverse reactions or were hard to read.®TB Gold in-tube-test  was used. One ml of venous blood was drawn into one tube pre-coated with synthetic peptide antigens and one tube without antigens (negative control sample) and transported the same day to the Norwegian Institute of Public Health for analysis. Samples were incubated, processed and stored in accordance with the manufacturer's instructions before harvested plasma was subjected to Enzyme-Linked Immunosorbent Assay (ELISA) analysis, including IFN-γ standard for quantification. The quality of all laboratory analysis and calculation of the results was controlled by using the accompanying QFT analysis software. A sample was considered positive if exceeding the standard cut-off value at 0.35 IU IFN-γ/ml. All positive results were confirmed by re-analysis of the same plasma sample before reported as positive. If it was not possible to reproduce a positive result, the QFT result was reported as non-conclusive and the participant excluded from the study.QuantiFERONData were entered into Excel locally and later validated at the Norwegian Institute of Public Health. Kappa-statistics, confidence intervals for single samples (Wilson's method) and unpaired samples (Newcombe's method) were calculated in Confidence Interval Analysis Software  . The othA total of 2963 asylum seekers, 18 years and older, arrived in Norway during the study period and 2813 (95%) of them entered Tanum reception centre. Of the 1000 participants enrolled, 88 (8.8%) were later excluded due to: withdrawal (1), missing TST-results (5), no QFT result due to technical routine error during blood sampling or analyses (47), non-reproducible positive result on the first plasma sample (34) and in one case the first analysis showed too high background value in the negative control sample, while the second analysis was positive. There was not enough plasma for a new analysis and this case (1) was excluded due to lack of confirmation of the positive result. Only the exclusion of the latter 35 cases is attributable to the performance of the QFT assay, and many of these cases had results clustered at or very near the IFN-γ cut-off value (0.35 U/ml). Their TST indurations were in the range of 0–22 mm (median 7 mm). This left 912 participants with valid QFT- and TST results. The study group included significantly more men and fewer persons from Europe compared to all arriving asylum seekers  participants tested positive with TST (≥ 6 mm) while 264 (29%) tested positive with QFT. Among the TST positive participants 50% were QFT negative, whereas 7% of the TST negative participants (< 6 mm) tested positive with QFT. Positive TST and QFT tests were most common among African participants (63% and 43%). European participants more often tested positive on TST (52%) than Asian participants (39%), while QFT-results were similar among European and Asian participants, 19% vs 20% respectively. Positive QFT-results were seen with all sizes of TST indurations, but the proportion of positive QFT tests increased with increasing induration size Figure  and 1b. Overall kappa-values for agreement between QFT and TST were fair to moderate depending on the chosen cut-off values for TST, and they were highest with TST cut-off ≥ 10 mm Table . The agrPredictors of a positive QFT-result in multivariate regression analysis were age, origin and exposure. With TST ≥ 15 mm as outcome variable gender, age, and origin were predictors Table  and 3b. If QFT is implemented in routine screening the number in need of referral could be reduced by 43% compared with referral based only on a positive TST ≥ 6 mm (50% TST positives compared to 29% QFT positives)  we found that 26% (37 out of 141) had a negative QFT test. High proportions of discrepant results in this group has also been observed in other studies ,30,31. AThe multivariate analysis showed similar significant risk factors for positive QFT and TST >15 mm. Country of origin and increase in age were both significantly associated with both positive QFT and TST ≥15 mm, reflecting accumulated exposure time and background prevalence in country of origin. Exposure to an infectious case of tuberculosis was significantly related to QFT, but not to TST, as found in other studies -9,41-44.There is no gold standard test for latent infection. Both positive QFT results and TST>15 mm (independent of each other) have been suggested to accurately identify latent infection and also predict progression to active disease ,23,28 WeThe prevalence of latent infection among asylum seekers was high, both defined by TST (50%) and QFT (29%), and with the expected discordance between the two test methods. The increased specificity of QFT is a very important improvement in a screening context as the number of persons referred for preventive treatment would be reduced by 43% independent of whether the one- or two-step approach is implemented, compared to referral based on TST (≥ 6 mm) alone. All three screening approaches would identify a similar proportion (88 – 89%) of those defined as having latent infection, even though it would not be the same persons referred. As only 0.5% of the study group had active tuberculosis disease on arrival, identification of persons with latent infection for referral and assessment for preventive treatment remains a key objective of the screening on entry. Identification of active disease also relies on screening of symptoms and chest x-ray.The authors declare that they have no competing interests.BAW has been responsible for study design, planning, data collection, data analysis and interpretation of data and the writing process, FO has been responsible for the laboratory components of the study and has made substantial contributions to the writing process, GEK carried out laboratory work and contributed to interpretation of data, TM contributed to planning of the study, interpretation of data and input in the writing process, ASJ has contributed to study design, data collection and quality assessment of data, IH participated in data collection and analyses of results, BH contributed to statistical analyses of data and EH contributed to planning of the study, data interpretation and analyses of results as well as giving substantial input in the writing process. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The objective of this study was to determine if MTND2*LHON4917G (4917G), a specific non-synonymous polymorphism in the mitochondrial genome previously associated with neurodegenerative phenotypes, is associated with increased risk for age-related macular degeneration (AMD). A preliminary study of 393 individuals (293 cases and 100 controls) ascertained at Vanderbilt revealed an increased occurrence of 4917G in cases compared to controls . Since there was a significant age difference between cases and controls in this initial analysis, we extended the study by selecting Caucasian pairs matched at the exact age at examination. From the 1547 individuals in the Vanderbilt/Duke AMD population association study (including 157 in the preliminary study), we were able to match 560 (280 cases and 280 unaffected) on exact age at examination. This study population was genotyped for 4917G plus specific AMD-associated nuclear genome polymorphisms in CFH, LOC387715 and ApoE. Following adjustment for the listed nuclear genome polymorphisms, 4917G independently predicts the presence of AMD . In conclusion, a specific mitochondrial polymorphism previously implicated in other neurodegenerative phenotypes (4917G) appears to convey risk for AMD independent of recently discovered nuclear DNA polymorphisms. Age-related macular degeneration (AMD) is the leading cause of irreversible severe vision loss in Caucasians over the age of 50.Stable single nucleotide polymorphisms (SNPs) have emerged in the mitochondrial genome over the past 150,000 years. This genetic variation results in distinctive sets of human mitochondrial electron transport chains with different capacities for energy production, free radical generation and apoptosis. Variations in the mitochondrial genome have been associated with neurodegenerative disorders, including Parkinson disease, Alzheimer disease, Friedrich's ataxia, and amytrophic lateral sclerosis.AMD patients and unrelated controls were ascertained at both Vanderbilt University Medical Center (VUMC) and Duke University Medical Center (DUMC). The study protocol had approval by the Institutional Review Boards at both institutions and followed the tenets of the Declaration of Helsinki. Informed consent was obtained from all participants.The preliminary study group consisted of individuals ascertained exclusively at Vanderbilt (N = 393). Following the preliminary study, the study population was expanded to include all N = 1547 individuals in the combined Vanderbilt/Duke AMD study population. From this group, which included all individuals in the preliminary study, we matched on the exact age (in years) at the time of the examination in order to negate any confounding effect from the difference in age between cases and controls. We were able to successfully match on exact age for N = 560 individuals (280 cases and 280 controls). Of these 560 individuals, 157 were in the preliminary study (83 cases and 74 controls). The age-matched group was an attempt at the discovery of an association independent of other nuclear genetic confounders and free of confounding from differences in age at examination. In no way did this study represent a validation of the initial finding since individuals are in both groups. The preliminary data (N = 393) is presented simply to report what actually occurred.A standard protocol was used throughout ascertainment at both locations. Taqman assay. The mtDNA 4917G snp was detected by using a MGB Eclipse Probe because of a polymorphism at the position 4918 . The 4917 forward primer was CAACTGCCTGCTA*TGATGGAT and the 4917 reverse primer was GGCCTGCTTCTTCTCACATGACA . The FAM probe for the wild type 4917 allele was TTACGA*TT*A*GT*GN*GG and the TET probe for the 4917G allele was TTACGA*CTA*GT*GN*GG  Genotypic data were analyzed using ABI Sequence Detection System version 2.1 software and confirmed by visual inspection of the plots. The genotyping for the specific CFH, LOC387715, and ApoE polymorphisms were carried out using ABI assays that have been described elsewhere. DNA was isolated from whole blood using PUREGENE . Genotyping was performed with the ABI PRISM 7900HT Sequence Detection System  using the 5′nuclease allelic discrimination 2 test. The 4917G genotype frequencies were calculated as the proportion of cases or controls that carried A or G alleles. This polymorphism is generally homoplasmic . Tests for statistical significance were two-sided with an alpha level of 0.05. Multivariable logistic regression was used to assess the relationship between independent variables and the outcome. Effect size for the association is measured as odds ratio (OR) with 95% confidence intervals. Statistical Analysis Software version 9.1  was used for all statistical analyses.Mitochondrial 4917G genotype frequencies were compared between cases and controls using a χThe initial study population consisted of 393 individuals (293 cases and 100 controls) ascertained at Vanderbilt University Medical Center. The clinical characteristics of these individuals are presented in Following this preliminary study, we selected 280 tightly age-matched Caucasian pairs from the existing large AMD study population ascertained at Vanderbilt and Duke University. The age at exam of both groups was 70.0 years±7.6. The univariate analysis of both clinical and genotype data in this population is presented in In this study, we observed that 4917G, a non-synonymous mitochondrial DNA polymorphism associated with mitochondrial haplogroup T, appears to confer an increased risk for AMD. This variation in the mitochondrial genome was an independent predictor of AMD following multivariate adjustment for well-known nuclear genetic factors. This observation should not be surprising, since mitochondria are vitally important in free radical production, apoptosis and cellular energy production. While this study provides epidemiologic evidence of relationship between mtDNA 4917G and susceptibility to developing AMD, the biological mechanism underlying this association remains to be elucidated. The nonconservative amino acid change resulting from the 4917G polymorphism in the ND2 gene may increase the rate of electron leakage from the mitochondrial electron transport chain. As a direct consequence, reactive oxygen species (ROS) production may increase and result in damage to many important biomolecules. Genetic factors other than 4917G are clearly important in the development and progression of AMD. Our analysis of other established genetic risk factors for AMD was consistent with the published literature. However, 4917G remained an independent predictor of AMD following adjustment for these known risk factors. It is possible that the 4917G mtDNA polymorphism is in linkage disequilibrium with more important causative polymorphisms. Of course, replication of these results in other populations of suitable sample size and power will provide even more convincing evidence for the role this mtDNA polymorphism plays in AMD. Epistatic interactions between individual loci within the mitochondrial genome as well as nuclear-mitochondrial gene interactions, for instance perhaps involving LOC387715, will be investigated further in larger studies. In summary, this study provides new evidence that variation in the mitochondrial genome contributes to susceptibility to AMD. The magnitude of the risk associated with the haplogroup T suggests that it may be an important new risk factor for the development of AMD. Future studies involving AMD will need to take into account the distribution of 4917G in their study populations. As we move forward with genome-wide association studies of AMD that span the 6,000,000,000 base pairs of the human diploid nuclear genome, it is worth noting again that humans have two genomes.


The reporting of observational studies is often of insufficient quality. Poor reporting hampers the assessment of the strengths and weaknesses of a study and the generalisability of its results. Taking into account empirical evidence and theoretical considerations, a group of methodologists, researchers, and editors developed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) recommendations to improve the quality of reporting of observational studies. The STROBE Statement consists of a checklist of 22 items, which relate to the title, abstract, introduction, methods, results and discussion sections of articles. Eighteen items are common to cohort studies, case-control studies and cross-sectional studies and four are specific to each of the three study designs. The STROBE Statement provides guidance to authors about how to improve the reporting of observational studies and facilitates critical appraisal and interpretation of studies by reviewers, journal editors and readers. This explanatory and elaboration document is intended to enhance the use, understanding, and dissemination of the STROBE Statement. The meaning and rationale for each checklist item are presented. For each item, one or several published examples and, where possible, references to relevant empirical studies and methodological literature are provided. Examples of useful flow diagrams are also included. The STROBE Statement, this document, and the associated Web site ( In this explanatory and elaboration document Mattias Egger and colleagues provide the meaning and rationale of each checklist item on the STROBE Statement. Rational health care practices require knowledge about the aetiology and pathogenesis, diagnosis, prognosis and treatment of diseases. Randomised trials provide valuable evidence about treatments and other interventions. However, much of clinical or public health knowledge comes from observational research . About nReporting of observational research is often not detailed and clear enough to assess the strengths and weaknesses of the investigation ,5. To imSTROBE provides general reporting recommendations for descriptive observational studies and studies that investigate associations between exposures and health outcomes. STROBE addresses the three main types of observational studies: cohort, case-control and cross-sectional studies. Authors use diverse terminology to describe these study designs. For instance, ‘follow-up study' and ‘longitudinal study' are used as synonyms for ‘cohort study', and ‘prevalence study' as synonymous with ‘cross-sectional study'. We chose the present terminology because it is in common use. Unfortunately, terminology is often used incorrectly  or impreObservational studies serve a wide range of purposes: from reporting a first hint of a potential cause of a disease, to verifying the magnitude of previously reported associations. Ideas for studies may arise from clinical observations or from biologic insight. Ideas may also arise from informal looks at data that lead to further explorations. Like a clinician who has seen thousands of patients, and notes one that strikes her attention, the researcher may note something special in the data. Adjusting for multiple looks at the data may not be possible or desirable , but furThis paper is linked to the shorter STROBE paper that introduced the items of the checklist in several journals , and forSTROBE recommendations do not specifically address topics such as genetic linkage studies, infectious disease modelling or case reports and case series ,12. As mWe now discuss and explain the 22 items in the STROBE checklist , and giv“Leukaemia incidence among workers in the shoe and boot manufacturing industry: a case-control study” .Readers should be able to easily identify the design that was used from the title or abstract. An explicit, commonly used term for the study design also helps ensure correct indexing of articles in electronic databases ,20.Background: The expected survival of HIV-infected patients is of major public health interest.“Objective: To estimate survival time and age-specific mortality rates of an HIV-infected population compared with that of the general population.Design: Population-based cohort study.Setting: All HIV-infected persons receiving care in Denmark from 1995 to 2005.Patients: Each member of the nationwide Danish HIV Cohort Study was matched with as many as 99 persons from the general population according to sex, date of birth, and municipality of residence.Measurements: The authors computed Kaplan–Meier life tables with age as the time scale to estimate survival from age 25 years. Patients with HIV infection and corresponding persons from the general population were observed from the date of the patient's HIV diagnosis until death, emigration, or 1 May 2005.Results: 3990 HIV-infected patients and 379 872 persons from the general population were included in the study, yielding 22 744  and 2 689 287  person-years of observation. Three percent of participants were lost to follow-up. From age 25 years, the median survival was 19.9 years  among patients with HIV infection and 51.1 years  among the general population. For HIV-infected patients, survival increased to 32.5 years  during the 2000 to 2005 period. In the subgroup that excluded persons with known hepatitis C coinfection (16%), median survival was 38.9 years  during this same period. The relative mortality rates for patients with HIV infection compared with those for the general population decreased with increasing age, whereas the excess mortality rate increased with increasing age.Limitations: The observed mortality rates are assumed to apply beyond the current maximum observation time of 10 years.Conclusions: The estimated median survival is more than 35 years for a young person diagnosed with HIV infection in the late highly active antiretroviral therapy era. However, an ongoing effort is still needed to further reduce mortality rates for these persons compared with the general population” . Of. Of94]. In an empirical study, only 93 of 169 articles (55%) reporting adjustment for confounding clearly stated how continuous and multi-category variables were entered into the statistical model . Anothera−b−, a−+, a+bb−, and +b+a) was redefined for sex and a dichotomous exposure of interest where −a and −b denote absence of exposure. RR was calculated for each category after adjustment for age. An interaction effect is defined as departure from additivity of absolute effects, and excess RR caused by interaction (RERI) was calculated:+b+a) denotes RR among those exposed to both factors where RR(a−b−) is used as reference category (RR = 1.0). Ninety-five percent CIs were calculated as proposed by Hosmer and Lemeshow. RERI of 0 means no interaction”  is estimated to result in five (95% CI 3–7) additional breast cancers per 1000 users of oestrogen-only preparations and 19 (15–23) additional cancers per 1000 users of oestrogen-progestagen combinations” .The results from studies examining the association between an exposure and a disease are commonly reported in relative terms, as ratios of risks, rates or odds see . RelativWidely used statistical models, including logistic  and propIn many circumstances, the absolute risk associated with an exposure is of greater interest than the relative risk. For example, if the focus is on adverse effects of a drug, one will want to know the number of additional cases per unit time of use . The example gives the additional number of breast cancer cases per 1000 women who used hormone-replacement therapy for 10 years . MeasureA recent survey of abstracts of 222 articles published in leading medical journals found that in 62% of abstracts of randomised trials including a ratio measure absolute risks were given, but only in 21% of abstracts of cohort studies . A free Observational studies may be solely done to describe the magnitude and distribution of a health problem in the population. They may examine the number of people who have a disease at a particular time , or that develop a disease over a defined period (incidence). The incidence may be expressed as the proportion of people developing the disease (cumulative incidence) or as a rate per person-time of follow-up (incidence rate). Specific terms are used to describe different incidences; amongst others, mortality rate, birth rate, attack rate, or case fatality rate. Similarly, terms like point prevalence and period, annual or lifetime prevalence are used to describe different types of prevalence .Other observational studies address cause-effect relationships. Their focus is the comparison of the risk, rate or prevalence of the event of interest between those exposed and those not exposed to the risk factor under investigation. These studies often estimate a ‘relative risk', which may stand for risk ratios (ratios of cumulative incidences) as well as rate ratios (ratios of incidence rates). In case-control studies only a fraction of the source population (the controls) are included. Results are expressed as the ratio of the odds of exposure among cases and controls. This odds ratio provides an estimate of the risk or rate ratio depending on the sampling of cases and controls see also  175,176176. The Expressing results both in relative and absolute terms may often be helpful. For example, in a study of male British doctors the incidence rate of death from lung cancer over 50 years of follow-up was 249 per 100,000 per year among smokers, compared to 17 per 100,000 per year among non-smokers: a rate ratio of 14.6 (249/17) . For coreffects, estimates of relative risk may be translated into suitable measures of absolute risk in order to gauge the possible impact of public health policies (see item 16c) [How much of the disease burden in a population could be prevented by eliminating an exposure? Global estimates have been published for smoking: according to one study 91% of all lung cancers, 40% of CHD and 33% of all deaths among men in 2000 were attributed to smoking . The poptem 16c) . Howevertem 16c) . Care isIn addition to the main analysis other analyses are often done in observational studies. They may address specific subgroups, the potential interaction between risk factors, the calculation of attributable risks, or use alternative definitions of study variables in sensitivity analyses.There is debate about the dangers associated with subgroup analyses, and multiplicity of analyses in general ,104. In A third area of debate is how joint effects and interactions between risk factors should be evaluated: on additive or multiplicative scales, or should the scale be determined by the statistical model that fits best . Judgement is needed regarding the level of reporting of such analyses. If many sensitivity analyses were performed, it may be impractical to present detailed findings for them all. It may sometimes be sufficient to report that sensitivity analyses were carried out and that they were consistent with the main results presented. Detailed presentation is more appropriate if the issue investigated is of major concern, or if effect estimates vary considerably ,186.Pocock and colleagues found that 43 out of 73 articles reporting observational studies contained subgroup analyses. The majority claimed differences across groups but only eight articles reported a formal evaluation of interaction (see item 12b) [Interaction exists when the association of an exposure with the risk of disease differs in the presence of another exposure. One problem in evaluating and reporting interactions is that the effect of an exposure can be measured in two ways: as a relative risk (or rate ratio) or as a risk difference (or rate difference). The use of the relative risk leads to a multiplicative model, while the use of the risk difference corresponds to an additive model ,188. A dRegardless of the model choice, the main objective is to understand how the joint effect of two exposures differs from their separate effects (in the absence of the other exposure). The Human Genomic Epidemiology Network (HuGENet) proposed a lay-out for transparent presentation of separate and joint effects that permits evaluation of different types of interaction . Data frA difficulty is that some study designs, such as case-control studies, and several statistical models, such as logistic or Cox regression models, estimate relative risks (or rate ratios) and intrinsically lead to multiplicative modelling. In these instances, relative risks can be translated to an additive scale. In example 1 of item 17, the separate odds ratios are 3.7 and 6.9; the joint odds ratio is 34.7. When these data are analysed under a multiplicative model, a joint odds ratio of 25.7 is expected (3.7 × 6.9). The observed joint effect of 34.7 is 1.4 times greater than expected on a multiplicative scale (34.7/25.7). This quantity (1.4) is the odds ratio of the multiplicative interaction. It would be equal to the antilog of the estimated interaction coefficient from a logistic regression model. Under an additive model the joint odds ratio is expected to be 9.6 (3.7 + 6.9 – 1). The observed joint effect departs strongly from additivity: the difference is 25.1 (34.7 – 9.6). When odds ratios are interpreted as relative risks (or rate ratios), the latter quantity (25.1) is the Relative Excess Risk from Interaction (RERI) . This caAnnals of Internal Medicine [The discussion section addresses the central issues of validity and meaning of the study . SurveysMedicine  recommenMedicine ,194. TheMedicine ,198. We “We hypothesized that ethnic minority status would be associated with higher levels of cardiovascular disease (CVD) risk factors, but that the associations would be explained substantially by socioeconomic status (SES). Our hypothesis was not confirmed. After adjustment for age and SES, highly significant differences in body mass index, blood pressure, diabetes, and physical inactivity remained between white women and both black and Mexican American women. In addition, we found large differences in CVD risk factors by SES, a finding that illustrates the high-risk status of both ethnic minority women as well as white women with low SES“ .It is good practice to begin the discussion with a short summary of the main findings of the study. The short summary reminds readers of the main findings and may help them assess whether the subsequent interpretation and implications offered by the authors are supported by the findings.“Since the prevalence of counseling increases with increasing levels of obesity, our estimates may overestimate the true prevalence. Telephone surveys also may overestimate the true prevalence of counseling. Although persons without telephones have similar levels of overweight as persons with telephones, persons without telephones tend to be less educated, a factor associated with lower levels of counseling in our study. Also, of concern is the potential bias caused by those who refused to participate as well as those who refused to respond to questions about weight. Furthermore, because data were collected cross-sectionally, we cannot infer that counseling preceded a patient's attempt to lose weight” .The identification and discussion of the limitations of a study are an essential part of scientific reporting. It is important not only to identify the sources of bias and confounding that could have affected results, but also to discuss the relative importance of different biases, including the likely direction and magnitude of any potential bias  and the measurement of exposures, confounders and outcomes (item 8). The inability to precisely measure true values of an exposure tends to result in bias towards unity: the less precisely a risk factor is measured, the greater the bias. This effect has been described as ‘attenuation' ,202, or The Lancet found that important weaknesses of the study were reported by the investigators in the survey questionnaires, but not in the published article [When discussing limitations, authors may compare the study being presented with other studies in the literature in terms of validity, generalisability and precision. In this approach, each study can be viewed as contribution to the literature, not as a stand-alone basis for inference and action . Surpris article .“Any explanation for an association between death from myocardial infarction and use of second generation oral contraceptives must be conjectural. There is no published evidence to suggest a direct biologic mechanism, and there are no other epidemiologic studies with relevant results. (…) The increase in absolute risk is very small and probably applies predominantly to smokers. Due to the lack of corroborative evidence, and because the analysis is based on relatively small numbers, more evidence on the subject is needed. We would not recommend any change in prescribing practice on the strength of these results” .The heart of the discussion section is the interpretation of a study's results. Over-interpretation is common and human: even when we try hard to give an objective assessment, reviewers often rightly point out that we went too far in some respects. When interpreting results, authors should consider the nature of the study on the discovery to verification continuum and potential sources of bias, including loss to follow-up and non-participation . Due consideration should be given to confounding (item 16a), the results of relevant sensitivity analyses, and to the issue of multiplicity and subgroup analyses (item 17). Authors should also consider residual confounding due to unmeasured variables or imprecise measurement of confounders. For example, socioeconomic status (SES) is associated with many health outcomes and often differs between groups being compared. Variables used to measure SES  are surrogates for other undefined and unmeasured exposures, and the true confounder will by definition be measured with error . AuthorsTo guide thinking and conclusions about causality, some may find criteria proposed by Bradford Hill in 1965 helpful . How str”How applicable are our estimates to other HIV-1-infected patients? This is an important question because the accuracy of prognostic models tends to be lower when applied to data other than those used to develop them. We addressed this issue by penalising model complexity, and by choosing models that generalised best to cohorts omitted from the estimation procedure. Our database included patients from many countries from Europe and North America, who were treated in different settings. The range of patients was broad: men and women, from teenagers to elderly people were included, and the major exposure categories were well represented. The severity of immunodeficiency at baseline ranged from not measureable to very severe, and viral load from undetectable to extremely high” .per se; the term is meaningful only with regard to clearly specified conditions [Generalisability, also called external validity or applicability, is the extent to which the results of a study can be applied to other circumstances . There inditions . Can resThe question of whether the results of a study have external validity is often a matter of judgment that depends on the study setting, the characteristics of the participants, the exposures examined, and the outcomes assessed. Thus, it is crucial that authors provide readers with adequate information about the setting and locations, eligibility criteria, the exposures and how they were measured, the definition of outcomes, and the period of recruitment and follow-up. The degree of non-participation and the proportion of unexposed participants in whom the outcome develops are also relevant. Knowledge of the absolute risk and prevalence of the exposure, which will often vary across populations, are helpful when applying results to other settings and populations see .Some journals require authors to disclose the presence or absence of financial and other conflicts of interest ,218. SevAuthors or funders may have conflicts of interest that influence any of the following: the design of the study ; choice The STROBE Statement aims to provide helpful recommendations for reporting observational studies in epidemiology. Good reporting reveals the strengths and weaknesses of a study and facilitates sound interpretation and application of study results. The STROBE Statement may also aid in planning observational studies, and guide peer reviewers and editors in their evaluation of manuscripts.We wrote this explanatory article to discuss the importance of transparent and complete reporting of observational studies, to explain the rationale behind the different items included in the checklist, and to give examples from published articles of what we consider good reporting. We hope that the material presented here will assist authors and editors in using STROBE.http://www.strobe-statement.org/) provides a forum for discussion and suggestions for improvements of the checklist, this explanatory document and information about the good reporting of epidemiological studies.We stress that STROBE and other recommendations on the reporting of research ,233,234 http://www.strobe-statement.org/ for current list). We invite other journals to adopt the STROBE Statement and contact us through our Web site to let us know. The journals publishing the STROBE recommendations provide open access. The STROBE Statement is therefore widely accessible to the biomedical community.Several journals ask authors to follow the STROBE Statement in their instructions to authors (see


The International Prognostic Index (IPI) is used to determine prognosis in diffuse large B-cell lymphoma (DLBCL). One of the determinants of IPI is the stage of disease with bone marrow involvement being classified as stage IV. For the IPI, involvement on bone marrow is traditionally defined on the basis of histology with ancillary investigations used only in difficult cases to aid histological diagnosis. This study aimed to determine the effect of the routine use of flow cytometry, immunohistochemistry and molecular studies in bone marrow staging upon the IPI.Bone marrow trephines of 156 histologically proven DLBCL cases at initial diagnosis were assessed on routine histology, and immunohistochemistry using two T-cell markers (CD45RO and CD3), two B-cell markers (CD20 and CD79a) and kappa and lambda light chains. Raw flow cytometry data on all samples were reanalysed and reinterpreted blindly. DNA extracted from archived paraffin-embedded trephine biopsy samples was used for immunoglobulin heavy chain and light chain gene rearrangement analysis. Using immunophenotyping (flow cytometry and immunohistochemistry), 30 (19.2%) cases were upstaged to stage IV. A further 8 (5.1%) cases were upstaged using molecular studies. A change in IPI was noted in 18 cases (11.5%) on immunophenotyping alone, and 22 (14.1%) cases on immunophenotyping and molecular testing. Comparison of two revised IPI models, 1) using immunophenotyping alone, and 2) using immunophenotyping with molecular studies, was performed with baseline IPI using a Cox regression model. It showed that the revised IPI model using immunophenotyping provides the best differentiation between the IPI categories.Improved bone marrow staging using flow cytometry and immunohistochemistry improves the predictive value of the IPI in patients with DLBCL and should be performed routinely in all cases. Diffuse large B-cell lymphoma (DLBCL) is defined by the World Health Organization (WHO) as a heterogeneous entity, encompassing morphologic and genetic variants, and variable clinical presentations and outcomes . It accoThe International Prognostic Index (IPI) is a standard clinical tool that is widely used to predict outcome for patients with aggressive Non-Hodgkin lymphoma (NHL), including DLBCL. It uses a number of clinical and laboratory markers present at the time of diagnosis to predict survival. Age > 60 years, stage III/IV disease defined by results of radiological investigations and bone marrow (BM) biopsy, elevated lactate dehydrogenase (LDH) level, Eastern Cooperative Oncology Group (ECOG) performance status ≥ 2 and more than one extra nodal site of disease, are scored 1 each, and depending on the final score ranging from 0-5, 4 prognostic categories are created. These are: low risk correlating with IPI of 0-1, low-intermediate risk with IPI of 2, high-intermediate risk with IPI of 3, and high risk with IPI of 4-5. Five year overall survivals range from 73% to 26%  was available in 150 patients. Thirty nine (26%), 35 (23%), 45 (30%), and 31 (21%) were found to have stage I, stage II, stage III and stage IV disease respectively. Baseline assessment of IPI was possible in 148 patients. Thirty seven (25%) had an IPI of ≤ 1 of which 14 (9.5%) had an IPI of 0, and 23 (15.5%) an IPI of 1. IPIs of 2 and 3 were noted in 36 (24.3%) and 46 (31.1%) cases respectively. Twenty nine (19.6%) cases had an IPI of ≥ 4 of which 22 (14.9%) and 7 (4.7%) and an IPI of 4 and 5 respectively. The mean baseline IPI of the patient cohort was 2.41 with a standard deviation of 1.3.Treatment data showed that of 152 patients on whom data was available, most were treated with anthracycline based regimens. One hundred and twenty nine patients 82.7%) were treated with Cyclophosphamide, Doxorubicin, Vincristine and Prednisolone (CHOP) % were tr. Two patBM biopsies are performed as a routine assessment for all cases with DLBCL at first diagnosis in our institution. All trephines are fixed in buffered formalin and acetic acid for 24 hours and then decalcified using 5% nitric acid. Samples are then embedded in paraffin and sections stained with Haematoxylin and Eosin (H&E), giemsa stain and silver impregnation for reticulin. Archived H&E, giemsa and reticulin preparations on the trephine biopsy were retrieved for review. The mean trephine length for the patient cohort was similar to our previous reports, 17.6 mm with a range of 8-36 mm and the mean number of levels on H&E sections were 3.7 (range 1-8).Two haematologists reviewed all slides blindly, and discrepant cases (n = 20) were resolved by consensus. Standardised criteria were used to classify trephine biopsy samples as positive, negative or indeterminate .Raw immunophenotypic data on all bone marrow biopsies was retrieved from laboratory records and re-analysed.Multiparametric flow cytometric analysis is performed in our laboratory, with marrow cells immunophenotypically labelled by direct four-colour immunofluorescence using a panel of antibodies .6 cells per tube is incubated with the monoclonal antibody at room temperature, then washed and resuspended in a solution of phosphate buffered saline and foetal calf serum. Isotypic controls used are IgG1 and IgG2.Red cells are lysed by incubation with ammonium chloride, and cells washed in phosphate buffered saline after centrifugation. A cell-suspension of 1 × 10Data acquisition is on a Becton-Dickinson flow cytometer with a minimum of 2000 lymphocytes counted in each sample. Bright CD45 fluorescent staining and intermediate side scatter are employed as the primary gating strategies to identify the lymphocyte population, and further gating is performed as required based on cell size or using back gating on CD19 positive events.Previously archived raw data were reanalysed, including blinded re-determination of the population of lymphocytes to be gated. Positive results on flow cytometry were defined as light chain clonal restriction with a kappa: lambda ratio of >3:1 or <0.3:1. PredomiImmunohistochemical analysis was performed on a Ventana Benchmark NexES machine. Sections from archived formalin-fixed decalcified paraffin-embedded (FFDPE) trephine biopsies were immunostained using the streptovidin-biotin method. The following monoclonal antibodies were used: CD3 , CD45RO , CD20 , CD79a , Kappa , and Lambda . All antibodies are validated and routinely used in our laboratory. CD20 and CD3 are reported to be sensitive at assigning lineage in diffuse aggressive NHL  and CD79Features used to define involvement on immunohistochemistry reflected standardised histology criteria. The presence of B-cell aggregates was classified as abnormal or malignant when there were large numbers of aggregates, the aggregates were large-sized, or contained disproportionate numbers of larger cells. Controls  were used to create a visual impression of normal amounts of background T and B-cells. Scattered small or large B-cells were classified positive only when the numbers were substantially increased as compared to controls. A conservative approach was adopted to avoid false positives. Discrepancies between the two pathologists were resolved consensually.Samples for molecular studies were obtained from formalin-fixed decalcified paraffin-embedded (FFDPE) trephine sections. DNA extraction was performed manually using the Roche High Pure PCR Template Preparation Kit from two 10-micron FFDPE trephine sections according to the manufacturer's instructions. To verify the integrity of the DNA extracted from the paraffin sections, and to validate results, all samples were amplified with the control master mix provided in the Immunoglobulin heavy chain (IgH) gene clonality kit from Invivo Scribe Technologies based on the BIOMED2 protocols . This is a multiplex PCR that targets multiple genes and generates a series of amplicons 100, 200, 300, 400 and 600 base pairs (bp) in length.A: VHFR1-JH] and FR3 [IGHC: VHFR3-JH] using the Invivo scribe kit based on the BIOMED2 protocols  and IgK variable and intragenic regions [IGKB: Vk-Kde]. The PCR reactions consisted of 45 μL of the FR1, FR3 or IGK master mix solution, 2.5 units of Amplitaq Gold  and 5 μl of template DNA (with an average template DNA concentration of 300-400 ng/μl). Thermo cycling was performed according to the kit protocol with no modifications on a Perkin Elmer 9600 thermocycler. Controls consisted of a positive DNA control, negative extraction control and negative PCR control. Water was used as a negative control in both cases.IgH gene rearrangement analysis was performed on all cases, targeting the conserved framework regions (FR) FR1 , rIPI1 category 3 = 7.6 , rIPI1 category 4/5 = 8.9 . The baseline IPI model and rIPI2 models were excluded from the regression model because they did not further contribute to explaining survival .Table To study the effect of treatment with Rituximab on survival, this too was considered, but not found to contribute significantly to the Cox regression model (p:0.96).In this study, we have shown a significant improvement in the predictive value of the IPI using ancillary staging investigations, particularly immunophenotyping, on the BM. By upstaging a proportion of cases, routine use of immunophenotyping provides better differentiation across the IPI prognostic categories. This has been confirmed using clinical outcomes in this study. These results validate the current guidelines that recommend incorporating immunophenotyping in routine staging and suggest the use of a new and more inclusive definition of BM involvement within the IPI.There have been several previous studies on the clinical role of ancillary investigations such as flow cytometry ,21-25, aOverall, multiparametric flow cytometry has been reported to be more sensitive than histology alone, and detection of flow cytometry positive cases have been reported in 3-11% of histologically negative cases, with rates in DLBCL varying from negligible to ~15% ,21-23,25Similarly, IHC is reported to detect marrow involvement in histologically negative cases in ~10-23% of cases depending on the histological diagnosis and the antibodies used ,27. ThisOverall, we found that use of immunophenotyping i.e. flow cytometry and immunohistochemistry in staging bone marrow biopsies upstages ~20-22% of patients with DLBCL. The two investigations complement each other. Flow cytometry is generally performed on aspirate samples and can be expected to add independent prognostic value. Immunohistochemistry, on the other hand, is performed on the trephine. Although it does not add independent prognostic value, it is a more sensitive technique than histology alone. This study shows the value of incorporating these tests as a routine rather than using them in histologically ambiguous cases only. Better differentiation into low-risk, low-intermediate, high-intermediate and high-risk IPI categories is obtained by using these tests on all staging bone marrows.There are several previous studies addressing the role of gene rearrangement (IgH/IgL) studies -31 in NHBesides the obvious advantage of availability of archived trephine biopsy tissue, the other reason for choosing to perform molecular staging on trephine biopsy rather than aspirate is that histological bone marrow involvement is noted more commonly on trephine biopsies. This has been demonstrated in previous studies  and largThere are several limitations of our study. This is a small retrospective study in DLBCL cases at initial diagnosis. As only a proportion of cases were treated with Rituximab in this study, we used multivariate analysis to demonstrate that it was not a significant confounding factor in our analysis. However, we acknowledge a prospective study may be required to confirm that the results are valid in Rituximab treated patients. The other major limitation is the use of archived rather than fresh trephine tissue for molecular staging due to the logistics of obtaining fresh trephine tissue in our centre. We acknowledge that we may have been able to demonstrate improvement in the prognostic significance of the IPI using gene rearrangement studies if fresh tissue had been analysed.Despite the limitations of our study, we were able to demonstrate an improvement in the prognostic significance of the IPI by use of simple, relatively inexpensive and readily available staging investigations such as flow cytometry and IHC. Our results suggest that a large prospective study is warranted to assess the impact of staging investigations on the IPI in a more homogenously treated DLBCL population.• The predictive value of the IPI can be improved significantly by the routine use of immunophenotyping on staging bone marrow biopsy.• Immunophenotyping i.e. flow cytometry and immunohistochemistry should be recommended as routine investigations on all bone marrows at initial diagnosis, as the detection of occult disease in morphologically normal marrow affects clinical outcome in DLBCL.• In this study, molecular analysis did not further contribute in improving the prognostic significance of the IPI. This is likely to have been due to technical limitations.• Larger prospective studies are warranted to assess the impact of staging investigations including gene rearrangement studies on the IPI in a more homogenously treated DLBCL population.BM: bone marrow; CT: computed tomography; DLBCL: diffuse large B-cell lymphoma; ECOG: eastern cooperative oncology group; FISH: fluorescent in-situ hybridisation; FR: framework; IG: immunoglobulin; IPI: International prognostic index; LDH: lactate dehydrogenase; NHL: Non Hodgkin Lymphoma; PET: positron emission tomography; rIPI: revised International prognostic index; WHO: World Health Organization.The authors declare that they have no competing interests.DT: project design, reporting histology, flow cytometry and immunohistochemistry. Performing all molecular analyses and interpreting results. Data entry and basic statistical analysis. Writing the paper. BS: input into project design, help set up database, advice on basic statistical analysis and performance of survival analyses. Input into and final approval of paper. JD: input into project design, designing standardised reporting format for immunohistochemistry, and blinded review of immunohistochemistry. Input into and final approval of paper. AM: reporting of histology slides, input into and approval of final paper.


A major goal of aging research is to identify interventions that prolong                        lifespan in distantly related organisms.  In recent years, genetic studies                        in both nematodes and rodents have reported that moderate inactivation of                        genes important for mitochondrial electron transport chain (ETC) function                        can promote longevity.  We performed an RNAi screen to probe the role of                        ETC components in modulating lifespan in the fruit flyDrosophila                        melanogaster.  In this Research Perspective, we discuss our findings                        and how they may relate to similar studies in worms and mice.                 Aging is characterized by a general decline in                        physiological function that leads to morbidity and eventually, mortality.   In recent years, significant progress has been made                        in our understanding of the nature of these changes at the molecular level.                         Due to the descriptive nature of these studies however,it has proven difficult to infer the relative                        importance of the many processes that contribute to aging.  Alterations in mitochondrial energy metabolism may be                        particularly important because it is central to metabolism a vital process that                        affects every aspect of cellular function.  Moreover, genetic studies in a                        number of model organisms have reported that inactivation of genes important                        for mitochondrial function can have profound effects on lifespan.                                Drosophila and how they relate to studies in other model                        organisms, including mammals.                                In this Perspective, we discuss our recent findings in                        the fruit fly Drosophila aging is also associated with changes in mitochondrial                            structure [Defects in mitochondrial electron transport chain                                                        as they aged in an effort to obtain a consensus aging transcriptsome. In both                            species, there was a significant decrease in a large set of genes involved in                            ATP synthesis and mitochondrial respiration [Another possible explanation behind the age-related                            decline in ETC function is the decline in expression of nuclear-encoded                            components of the ETC.  In one study, the                            authors compared microarray data between two organisms  can produce large effects on lifespan.  Although                            short-lived mutants are often considered less informative than long-lived mutants                            as they may result from novel pathologies unrelated to normal aging, the mev-1                            mutant may be a notable exception.  This short-lived mutant carries a mutation                            in subunit C of mitochondrial complex II [The link between mitochondrial energy metabolism and                            longevity has been most extensively studied in the nematode mplex II  and dispmplex II  which hamplex II .       isp-1) of                            mitochondrial complex III [clk-1 mutant lacks an enzyme required in the biosynthesis of                            ubiquinone, also known as coenzyme Q, an important electron acceptor for both                            complex I- and II-dependent respiration [One of the first demonstrations that impaired ETC                            function could increase lifespan was the isolation and characterization of a                            long-lived mutant with a mutation in the iron sulfur protein  results in very severe shortening of lifespan in both flies [sod-2                            deficiency in C. elegans has been reported to increase longevity in one                            study [When interpreting these findings, it may                            prove important to consider certain aspects of nematode ecology and physiology ,24.  Worth flies  and miceth flies .  In con   study  and have   study .  TherefDrosophila mutant with a defect in the iron-sulfur                            subunit (sdhB) of complex II was the first study to directly                            investigate the effects of decreased ETC function on fly longevity [mev-1) deficiency in                            worms.  Specifically, our biochemical data indicate that SDHB is critical in                            preventing electron leakage from complex II, so that mutant animals suffer from                            increased oxidative stress and, as a result, are highly sensitive to oxygen and                            die rapidly [The isolation and                            characterization of a ongevity .        rapidly .  The faDrosophila RNAi transgenes [C. elegans.                                        By happy coincidence the generation of a genome-wide                            library of ansgenes  was repoansgenes .  Not  ansgenes .   A majShortly after publication of the results of the RNAi                            screen, we reported that genetic and pharmacological treatments that target                            complex V affect lifespan in a nutrient-dependent manner .  In ansdhD die at early embryonic stages, while                            heterozygotes display complex II deficiency without alterations in body weight                            or major physiological dysfunction.  More recently, it was reported that                            inactivation of the Ndufs4 gene, which encodes an 18 kDa subunit of complex I,                            leads to an early-onset (5 weeks) encephalomyopathy [In humans, mitochondrial defects have been implicated                            in a wide range of life-shortening degenerative diseases ,37.  Howmyopathy .       There have also been a number of reports indicating                            that certain genetic manipulations that impair ETC function can protect against                            oxidative stress, neurodegeneration, obesity, and diabetes, and prolong                            longevity in mice.  For example, tissue-specific deletion of apoptosis inducing                            factor (AIF) leads to reduced ETC function and was shown to protect mice                            against both diabetes and obesity ,41.  RedTaken together, the work in flies, mice and the large                            number of long-lived ETC mutant worms clearly demonstrates that decreased                            expression of certain ETC genes is an evolutionarily conserved mode of lifespan                            extension.  However, the underlying mechanisms remain poorly understood.  Our                            own work indicates that ETC gene manipulations that prolong lifespan are not                            necessarily associated with obvious energetic or physiological trade-offs.  We                            identified five ETC gene hypomorphs associated with increased longevity.  However, only                            two of the ETC gene knock-downs conferred detectable decreases in the abundance                            of fully assembled respiratory complexes.  In addition, none of the five ETC                            gene perturbations resulted in a decrease in ATP levels.   An important next                            step will be a careful and detailed analysis of different aspects of                            respiratory chain function in long-lived flies with reduced expression of ETC                            genes.                                        Increased lifespan conferred byRNAi                            of mitochondrial respiratory components is not limited to nematodes but is also                            true for flies .   In adA challenge                            that we find particularly pressing is to reconcile our results with the large                            number of studies that report an age-related decline in ETC gene expression and                            activity.  If ETC activity decreases as a function of age, then it appears                            counterintuitive that RNAi knock-down                            of ETC genes would promote                            longevity.  In contrast, we might speculate that strategies to increase                            respiratory chain function may prove effective in retarding the aging process.                             In yeast, manipulations that increase respiration are associated with increased                            longevity ,47. Crit


Glucose-6-Phosphate dehydrogenase (G6PD) is a key enzyme of the pentose monophosphate pathway, and its deficiency is the most common inherited enzymopathy worldwide. G6PD deficiency is common among Iraqis, including those of the Kurdish ethnic group, however no study of significance has ever addressed the molecular basis of this disorder in this population. The aim of this study is to determine the prevalence of this enzymopathy and its molecular basis among Iraqi Kurds.A total of 580 healthy male Kurdish Iraqis randomly selected from a main regional premarital screening center in Northern Iraq were screened for G6PD deficiency using methemoglobin reduction test. The results were confirmed by quantitative enzyme assay for the cases that showed G6PD deficiency. DNA analysis was performed on 115 G6PD deficient subjects, 50 from the premarital screening group and 65 unrelated Kurdish male patients with documented acute hemolytic episodes due to G6PD deficiency. Analysis was performed using polymerase chain reaction/restriction fragment length polymorphism for five deficient molecular variants, namely G6PD Mediterranean (563 C→T), G6PD Chatham (1003 G→A), G6PD A- (202 G→A), G6PD Aures (143 T→C) and G6PD Cosenza (1376 G→C), as well as the silent 1311 (C→T) mutation.Among 580 random Iraqi male Kurds, 63 (10.9%) had documented G6PD deficiency. Molecular studies performed on a total of 115 G6PD deficient males revealed that 101 (87.8%) had the G6PD Mediterranean variant and 10 (8.7%) had the G6PD Chatham variant. No cases of G6PD A-, G6PD Aures or G6PD Cosenza were identified, leaving 4 cases (3.5%) uncharacterized. Further molecular screening revealed that the silent mutation 1311 was present in 93/95 of the Mediterranean and 1/10 of the Chatham cases.The current study revealed a high prevalence of G6PD deficiency among Iraqi Kurdish population of Northern Iraq with most cases being due to the G6PD Mediterranean and Chatham variants. These results are similar to those reported from neighboring Iran and Turkey and to lesser extent other Mediterranean countries. Glucose-6-Phosphate Dehydrogenase (G6PD) is a key enzyme in the pentose monophosphate pathway and provides the NADPH essential for a number of biosynthetic and detoxifying reactions. The gene encoding G6PD is located at Xq28, and its deficiency is the most common enzymopathy in humans. As an X-linked recessive trait, it is predominantly a disease of males affecting an estimated 400 million people worldwide . This deG6PD deficiency is prevalent in many countries in the Eastern Mediterranean Region including Iraq -8. Some Genetic studies including those of inherited blood disorders are very scarce on Kurds, and such studies may help explain the origin and spread of these disorders and may also help shed some light on the history of this ethnic group. No study of significance has addressed the frequency of G6PD deficiency in the Kurdish Iraqi population, and none has defined the deficient molecular variants among any group of Iraqis, despite the fact that favism is a commonly encountered health problem throughout the country.This study aims to: 1) determine the prevalence of G6PD deficiency in a random sample of healthy Iraqi Kurdish males attending a regional premarital screening center, and 2) define the molecular basis of G6PD deficiency among a sample of G6PD deficient males in the same region.The study was conducted in the period between July and December 2009. The subjects enrolled were males attending the Central laboratory in Dohuk, northern Iraq for routine premarital health screening. This laboratory serves a population of around one million of mainly ethnic Kurds, and is the only facility in this province that is authorized to provide government mandated premarital investigations. The latter currently include investigation for blood groups, HBsAg, HIV and carrier screening for specific hemoglobinopathies. The center receives on average around 30-40 couples per day. The study subjects were the male partners of the first 10 couples attending the center on every other working day throughout the study period.A 6 ml blood sample was collected in K2-EDTA tubes from each enrollee. The sample was used to screen for G6PD deficiency by methemoglobin reduction test . All thoAdditionally, 65 unrelated male patients who had documented hemolytic episodes associated with G6PD deficiency, were also recalled and had their G6PD deficient status reaffirmed by quantitative enzyme assay.All G6PD deficient individuals had their DNA extracted by a phenol-chloroform based method . The extInformed consent was taken form all enrollees or their guardians and the study was approved by the appropriate ethical committee at the Dohuk college of Medicine, Iraq.Statistical analysis utilized the Mann Whitney U test and Chi Squared test wherever appropriate. A p < 0.05 was considered significant.Among 580 Kurdish male premarital screening subjects, 63 individuals (10.9%) were found to be G6PD deficient by methemoglobin reduction test with all subsequently being confirmed as deficient by quantitative enzyme assay. Table Among the 63 G6PD deficient males detected, 50 responded to a recall for molecular characterization. Additionally, 65 previously diagnosed males with G6PD deficiency associated documented hemolytic episodes also underwent quantitative enzyme assays table  and moleThe silent mutation (1311 C→T) was documented in 93/95 (97.8%) of the Mediterranean deficient variants, in 1/10 of the Chatham cases and in one of the three uncharacterized variants.G6PD deficiency has long been recognized as a common inherited hematological disorder in Iraqis including Kurds ,8. KurdsEarlier limited studies on G6PD deficiency among Iraqi Kurdish males living in Baghdad (at the center of the country) have reported frequencies of 7.6-8.8% , which iThe predominance of the Mediterranean mutation 563 C→T) as demonstrated in the current study, has been documented by many studies from the Eastern Mediterranean countries, where it constitutes 54 to 91% of G6PD variants identified  seen in 8.7% of deficient individuals. G6PD Chatham, although first described in a patient of Asian Indian origin, is now recognized as one of the common variants worldwide . Table 5G6PD Cosenza (1376 G→C) was first described in Southern Italy  and has The African A-variant (202 G→A) was not detected among our sample. This is in contrast to an earlier report from central Iraq where it was reported in 4.3% of G6PD deficient subjects  and in sThis study has documented that the frequency of G6PD deficiency among the predominantly Kurdish population in Northern Iraq is 10.9%, which is higher than that reported from the neighboring Iranian Kurdish population. The study which is the first molecular study on G6PD deficient variants from Iraq documented that G6PD Mediterranean and Chatham constitute the large majority of the deficient variants which is similar to findings reported in surrounding populations. Further studies including larger number of patients, more diverse ethnic backgrounds and including screening for other deficient variants and DNA sequencing are needed to give a more comprehensive view of G6PD variants in Iraqis.The authors declare that they have no competing interests.NA, contributed the concept and design, part of the molecular studies, data analysis and drafting of the manuscript; AAE, contributed to the collection of data, larger part of the molecular work and analysis of data; JMSJ, contributed to the concept and design and part of the molecular work; SARJ, contributed to collection of data, performing hematological, enzyme assays and data analysis; HH, contributed to the analysis and interpretation of results, drafting and revision of the manuscript. All authors revised and approved the final submitted version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2326/10/6/prepub