The utility of serologic screening for celiac disease is still debatable. Evidence suggests that the disorder remains undetected even in the older population. It remains obscure whether screening makes good or harm in subjects with long-standing gluten ingestion. We evaluated whether older subjects benefit from active detection and subsequent gluten free dietary treatment of celiac disease.Thirty-five biopsy-proven patients aged over 50 years had been detected by serologic mass screening. We examined the disease history, dietary compliance, symptoms, quality of life and bone mineral density at baseline and 1-2 years after the commencement of a gluten-free diet. Symptoms were evaluated by gastrointestinal symptom rating scale and quality of life by psychological general well-being questionnaires. Small bowel biopsy, serology, laboratory parameters assessing malabsorption, and bone mineral density were investigated.Dietary compliance was good. The patients had initially low mean serum ferritin values indicating subclinical iron deficiency, which was restored by a gluten-free diet. Vitamin B12, vitamin D and erythrocyte folic acid levels increased significantly on diet. Celiac patients had a history of low-energy fractures more often than the background population, and the diet had a beneficial effect on bone mineral density. Alleviation in gastrointestinal symptoms was observed, even though the patients reported no or only subtle symptoms at diagnosis. Quality of life remained unchanged. Of all the cases, two thirds would have been diagnosed even without screening if the family history, fractures or concomitant autoimmune diseases had been taken carefully into account.Screen-detected patients benefited from a gluten-free diet. We encourage a high index of suspicion and active case-finding in celiac disease as an alternative to mass screening in older patients. Evidence suggests that the incidence of celiac disease increases with age ,2. PhysiOn the other hand, a lifelong gluten-free diet is restrictive and may also increase the burden of illness and impairs quality of life ,8. EspecIn this prospective study we evaluated the benefits of active serologic population-based mass screening for celiac disease and subsequent dietary treatment in subjects over 50 years of age. The aim was to establish whether celiac disease should be rigorously searched for in the older people.http://www.palmenia.helsinki.fi/ikihyva/InEnglish.html. In 2002, altogether 2815 subjects (66%) consented to participate and serum samples were drawn and stored at -20°C until used. Anti-tissue transglutaminase antibodies were tested from the stored sera in 2004, and 48 new seropositive cases were identified; their diagnostic work-up has been published elsewhere [We identified screen-detected new celiac disease patients enrolled from a cohort representing the older Finnish population. The original study population comprised 4272 randomly selected individuals born in the years 1946-50, 1936-40 and 1926-30, and was defined for a 10-year research project on ageing and well-being (Good Ageing in the Lahti region = GOAL) At baseline the patients were interviewed for their family history, associated disorders and symptoms of celiac disease. Nutritional condition and quality of life were evaluated. The patients were advised to start a gluten-free diet, and dietary counseling was carried out by a gastroenterologist and a dietician. To ensure dietary response and adherence to a gluten-free diet, an interview by the dietitian, small bowel biopsies and celiac disease antibodies were investigated after a median of one year; nutritional condition and quality of life were assessed after a median of two years on a gluten-free diet. The diet was considered strict when there were no signs of dietary transgressions upon the interview. Occasional gluten-free diet was defined as a gluten intake occurring less often than once in the month.Small bowel mucosal biopsy samples were taken by upper gastrointestinal endoscopy from the distal part of the duodenum. Three samples were paraffin-embedded, processed, stained with hematoxylin-eosin and studied under light microscopy. The villous height/crypt depth ratio (Vh/CrD) was calculated from well orientated biopsy specimens as previously described ; a ratioIgA-class tissue transglutaminase antibodies (TGA) were investigated by enzyme-linked immunosorbent assay  according to manufacturer's instructions; values ≥5.0 arbitrary units (U) were considered elevated. TGA-positive sera were further analyzed for IgA-class endomysial antibodies (EMA) by an indirect immunofluorescence method using human umbilical cord as substrate; a dilution of 1:5 or more was considered positive .The study patients were genotyped for HLA-DQB1*02, DQB1*0302 and DQA1*05 alleles using the DELFIA Coeliac Disease Hybridization Assay  according to manufacturer's instructions; DQB1*02 and DQA1*05 corresponding to associated alleles for HLA DQ2 and DQB1*0302 for HLA DQ8.The clinical symptoms were classified into three different subgroups: (i) no symptoms, (ii) subtle symptoms with occasionally one or more of the following: abdominal pain, flatulence, belching, loose stools, tiredness, joint pain or oral blisters, and (iii) classical symptoms with constant abdominal complaints, diarrhea or excessive weight loss. Abdominal complaints were additionally evaluated by the Gastrointestinal Symptom Rating Scale (GSRS), which denotes the total score derived from five different gastrointestinal symptoms; diarrhea, indigestion, constipation, abdominal pain and gastro-esophageal reflux; a higher score indicates more severe symptoms . Quality2 (kg/m2). Blood hemoglobin, erythrocyte folic acid levels, serum iron, ferritin, ionized calcium, phosphate and vitamin A, B-12, D-25 and E concentrations were measured using routine laboratory methods.Body mass index was computed as weight/heightBone mineral density was measured by dual-energy X-ray absorptiometry  in the spine (L1 to L4) and right femoral neck. Values were expressed as standard deviation (SD) scores, which compare individual values to the mean bone mineral density of sex-matched young adults (T score) or of the age- and sex-matched population (Z score). T scores above -1.0 SD represented normal values, scores between -1.0 and -2.4 osteopenia and scores ≤ -2.5 SD osteoporosis. When indicated, bisphosphonate medication together with supplementary calcium and vitamin D were recommended, since it was considered unethical to postpone medical treatment and wait for the effect of a gluten-free diet.The number of low-energy bone fractures was extracted from the questionnaires of the original GOAL study, equally in the total (n = 2815) series and in study subjects.The study was accepted by the Ethical Committee of Päijät-Häme Central Hospital, and written informed consent was obtained from all participants. Research is in compliance with the Helsinki Declaration.Data were given as means with 95% confidence intervals (CI) or medians with lower and upper quartiles and range when appropriate. Chi-square or Fisher's tests were used in cross tabulations Wilcoxon signed or paired rank test to compare changes within the study group. A p value < 0.05 was considered statistically significant. We calculated that, for the statistical power of 0.80 at a significance level of 0.05, 30 subjects would be sufficient. We estimated that the difference of > 0.5 in the Vh/CrD and that of 0.5 in GSRS were clinically significant ,14.All 35 new celiac disease patients identified by mass screening consented to participate Table . Celiac Thirty-two (91%) of the 35 screen-detected older celiac disease patients consented to start a gluten-free diet. Twenty-seven maintained a strict diet, and five had occasional transgressions less often than once in the month; three patients did not commence gluten-free diet.After one year on the diet, small bowel mucosal villous morphology improved and densities of IELs decreased statistically significantly in the 26 who agreed to undergo the follow-up biopsy Figure . In paraClinical symptoms resolved in 14 out of the 15 who reported symptoms at the time of diagnosis. One with initially subtle abdominal complaints developed diarrhea despite small bowel mucosal normalization on a strict gluten-free diet; colonoscopy showed no abnormal findings or inflammation. In accordance with the clinical history, alleviation in gastrointestinal symptoms was observed both by GSRS total score and by subscores, and was statistically significant, except in the case of constipation Table .12, D and E values improved significantly in those 32 who started the gluten-free diet , and vitamin D .Mean serum ferritin and vitamin BThe mean femoral Z-score improved significantly when patients had adhered to the gluten-free diet for two years; in the lumbar spine the improvement did not reach statistical significance Table . There wAn increasing number of patients with celiac disease will be diagnosed among the older people -3. The cOn the other hand, it is of crucial importance to know what the overall implications of the diagnosis are in older people. Mortality has not increased among older undiagnosed celiac disease patients  and the In these screen-detected patients an improvement in GSRS was evident under dietary treatment, both in total score and in virtually in all subscores, displaying an alleviation in gastrointestinal symptoms. The effect of the treatment on quality of life (PGWB) was not so evident, but it is of note that the diet did not worsen it. A comparable finding was obtained in our recent study where celiac patients detected by screening at risk groups were investigated . An imprA risk of low bone mineral density is possible in screen-detected apparently asymptomatic celiac disease patients . In the Compliance with a gluten-free diet does not seem to be a problem in older patients with celiac disease, as compliance rates have been more than 90% . AccordiNone of our celiac disease patients suffered from severe malabsorption syndrome, and they did not have refractory sprue or any other severe complications. Ten (29%) of these 35 screen-detected celiac patients had had relatives with celiac disease and 10 autoimmune diseases, which should both alert to celiac disease. Katz and associates  concludeScreen-detected, apparently asymptomatic older celiac disease patients may suffer from subclinical malabsorption, gastrointestinal symptoms or bone disease, which are alleviated during gluten-free dietary treatment. No deterioration in quality of life was seen in our series, and dietary compliance was excellent. Despite this, we consider that there is still insufficient evidence to advocate mass screening, until the costs and benefits of the approach have been thoroughly evaluated. Instead, as the majority of patients had a family history or associated conditions known to occur with celiac disease, we recommend active case finding in older people belonging to at-risk groups.The authors declare that they have no competing interests.AV, KK, LL, MM and PC conceived the study participated in the patient enrolment, data collection and study design: IK, ML and HP participated in the data collection and study design. RV and KL carried out the immunological studies and participated in the study design. 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/11/136/prepub


Introduction. To determine the value of a FDG-PET-CT scan in patients with locally advanced head and neck squamous cell carcinoma (HNSCC) prior to chemoradiotherapy. Materials and Methods. Consecutive patients with stage III or IV HNSCC who had undergone a staging FDG-PET-CT scan prior to chemoradiotherapy between August 2008 and April 2011 were included. Clinical details and conventional imaging (CT and/or MRI) were, retrospectively, reviewed, a TNM stage was assigned, and levels of cervical lymph node involvement were documented. This process was repeated with the addition of FDG-PET-CT. Radiotherapy plans were reviewed for patients with an alteration identified on TNM staging and/or nodal level identification with FDG-PET-CT and potential alterations in radiotherapy planning were documented. Results. 55 patients were included in the analysis. FDG-PET-CT altered the TNM stage in 17/55 (31%) of patients, upstaging disease in 11 (20%) and downstaging in 6 (11%); distant metastases were identified by FDG-PET-CT in 1 (2%) patient. FDG-PET-CT altered the lymph node levels identified in 22 patients (40%), upclassifying disease in 16 (29%) and downclassifying in 6 (11%). Radiotherapy plans were judged retrospectively to have been altered by FDG-PET-CT in 10 patients (18%). Conclusions. The use of FDG-PET-CT potentially impacts upon both treatment decisions and radiotherapy planning. Locoregional staging for head and neck squamous cell carcinoma (HNSCC) is performed using a combination of clinical examination, examination under anaesthetic and crosssectional imaging with computed tomography (CT) and/or magnetic resonance imaging (MRI). Anatomical imaging is fundamentally limited for detection of malignancy in nonnecrotic lymph nodes which fall below standard size criteria for example, short axis diameter of 10 mm . The risIn the modern era, organ preserving nonsurgical radiotherapy-based approaches to the management of locally advanced HNSCC are increasingly employed . Treatme18Fluoride-fluorodeoxyglucose positron emission tomography (FDG PET) is a widely used functional imaging technique in oncology. Tumour cells exhibit differential glucose uptake (the “Warburg effect”) as a basis of the identification of cancer [The radiation oncologist frequently has to make difficult judgements based on anatomical imaging to include or otherwise equivocal lymph nodes in the high-dose target volume. Functional imaging techniques offer the potential to provide complementary information to anatomical imaging with CT and MRI to aid in these decisions. f cancer . Increasf cancer . Limitatf cancer , 15, 16.f cancer . FDG PETf cancer . The usef cancer , whilst f cancer . Some stf cancer –23. At tThe aim of this study was to assess the utility of FDG PET-CT as an adjunct to conventional staging methods in patients with locally advanced HNSCC due to undergo primary nonsurgical treatment. The value of FDG PET-CT on identification of levels of nodal involvement was determined to assess whether FDG PET-CT can influence process of defining the radiotherapy target volume.Formal institutional review board approval was waived for this retrospective study. Consecutive patients between August 2008 and April 2011 who underwent a FDG PET-CT scan for head and neck cancer were obtained from an institutional database. Electronic case notes were used to identify patients who fulfilled the eligibility criteria for the study.Eligible patients fulfilled all of the following criteria.Histologically confirmed squamous cell carcinoma of the oropharynx, oral cavity, hypopharynx, larynx, or paranasal sinuses.Nasopharyngeal cancer was excluded.Reviewed by specialist head and neck multidisciplinary meeting.TNM stage III or IV prior to FDG PET-CT scan.Decision made prior to FDG PET-CT to proceed with radical nonsurgical treatment .FDG PET-CT performed prior to commencement of treatment for staging and/or as a baseline for future response assessment.Baseline demographics were obtained from review of electronic case notes .Conventional staging of locally advanced HNSCC was routinely performed by physical examination and neck palpation, fiberoptic endoscopy, examination under anaesthetic with biopsy where indicated, MRI or contrast-enhanced CT of head and neck region depending upon local protocols, and CT of the thorax. Results were routinely reviewed in a specialist head and neck MDT meeting and a TNM classification, based on all available clinical and radiological data, according to the American Joint Committee on Cancer TNM staging was assigned prior to the FDG PET-CT scan . The metContrast-enhanced CT was most commonly performed at our institution after referral from the regional oncology team on a 64-slice CT  using a contiguous 1 mm reconstruction following a bolus of 100 mL of iodinated contrast or a 16-slice CT  using the same acquisition parameters. The remaining contrast-enhanced CT scans were acquired at one of several referring hospitals on multislice CT systems using similar acquisition parameters.MRI examinations of the head and neck were most commonly performed at our institution using 1.5-Tesla systems . Scan limits were skull base to clavicles. Standard sequences included axial and coronal T1, axial T2 (fat sat) and coronal STIR, and axial and coronal T1 post-Gadolinium. Sagittal T1 post-Gadolinium was performed for tongue base tumours.18F-FDG. A silence protocol was employed in the uptake period following tracer injection to minimise physiological tracer activity within the head and neck region. The CT component was performed according to a standardised protocol (without the use of iodinated contrast medium) with the following settings: 140 kV; 80 mAs; tube rotation time 0.5 s per rotation; pitch 6; section thickness 3.75 mm (to match the PET section thickness). Patients maintained normal shallow respiration during the CT acquisition. Images were reconstructed using a standard OSEM algorithm with CT for attenuation correction. Both nonattenuation corrected and attenuation corrected datasets were reconstructed.FDG PET-CT examinations prior to June 2010 were performed on a 16-slice Discovery STE PET-CT scanner  and from June 2010 on a 64-slice Philips Gemini TF64 scanner . PET acquisition from skull vertex to upper thighs was performed 60 minutes after a 400 MBq dose of intravenous Imaging for each patient was retrieved from the institutional picture archiving and communications system (PACS). PET-CT imaging was reviewed on a specialised PET-CT workstation . Imaging was retrospectively reviewed by two experienced radiologists independently. Visual interpretation was qualitative, with regions with an FDG uptake above background mediastinal blood pool classed as positive. Any discrepancies were discussed in order to reach a consensus view. Clinical information and MRI and/or CT scans were reviewed by the same radiologists in conjunction with radiology reports. Standard size criteria  were useBased upon this data, any alterations in the TNM stage and/or levels of lymph node involvement between conventional imaging and PET-CT were documented.Management plan prior to FDG PET-CT staging was determined from electronic case notes and MDT records. Alterations in treatment modality/intent of treatment following FDG PET-CT were determined by retrospective review of records.During the early part of the study period, patients were treated with a conformal technique involving a parallel opposed pair ; this teIn general, using either technique, gross tumour volume was included within the high-dose volume, and areas deemed to be at lower risk were treated with prophylactic doses. In patients in whom a discrepancy was noted between the TNM staging and/or lymph node level identification, radiotherapy plans were reviewed to determine whether the lymph node was included in high-dose or prophylactic dose regions. A retrospective qualitative judgement was made as to whether FDG PET-CT altered had no impact or was ignored during the radiotherapy planning process. This was assessed by two experienced clinical oncologists independently, and any disagreements were discussed in order to reach a consensus view.Baseline characteristics and radiological findings were summarised using descriptive statistics.55 patients were eligible for inclusion in the study. Median age was 55 (range 28–75). 42/55 (76%) were male. Smoking, alcohol history, and performance status were recorded in 54 patients; 23 were current smokers, 10 exsmokers, and 21 had never smoked. 13 were classed as having a heavy alcohol intake (at least 50 units/week), 36 drank some alcohol (<50 units/week), and 5 did not drink alcohol. World Health Association (WHO) performance status was recorded in 54 patients: WHO PS 0,1,2 in 31, 16, and 7 patients, respectively.Tumour site, subsite, stage prior to FDG PET-CT, histology, and intended management plan prior to FDG PET-CT are shown in The median time between CT or MRI staging of the head and neck and the FDG PET-CT scan was 17.5 days (range 4–42). The median maximum SUV of the primary tumour was 13.1 (range 5.3–44) and for lymph nodes 8.7 (range 5.3-44). TNM stage at FDG PET-CT was discordant with staging utilising clinical information and CT and/or MRI in 17 (31%) of patients. The changes are summarised in The involvement of neck lymph node levels was determined utilising clinical information and CT and/or MRI imaging. There was discordance in lymph node level involvement on FDG PET-CT and conventional imaging in 22 of 55 (40%) patients . In both cases ultrasound-guided biopsies demonstrated reactive changes only, and there has been no clinical progression of the axillary lymph node after clinical followup of 14 and 8 months, respectively. Therefore, in these 2 cases, FDG PET-CT generated a false positive result outside of the head and neck region that had required further investigation.Advances in the ability to accurately identify all sites of disease underpin attempts to improve radiotherapy outcomes for HNSCC. Purely anatomical imaging with CT and MRI is inherently limited in failing to detect nonenlarged malignant lymph nodes. Functional imaging techniques, such as integrated FDG PET-CT, are not limited in the same manner, and their incorporation into existing staging protocols is highly attractive. FDG PET-CT could be used in the radiotherapy planning process to identify the target lymph nodes and/or to be used to contour target tissue. The delineation of target lymph nodes using FDG PET is highly dependent upon the methodology of PET segmentation which remains highly controversial , 27.Evidence with regard to the ability of FDG PET-CT to improve upon the staging of cervical lymph node levels is based upon multiple retrospective and prospective series. Kyzas et al.  performeA series of studies have explored whether the use of FDG PET-CT has any impact upon clinical management of HNSCC. Several series have now demonstrated the impact of FDG PET on management decisions , 31–36. It is important to note that within almost all of the studies in the Kyzas et al.  meta-anaOur series of 55 patients differs from other reported series in that a decision to proceed with primary nonsurgical had been made prior to the FDG PET-CT scan and that patients were restricted to stage III/IV HNSCC. FDG PET-CT was performed both for staging and to provide a baseline for future response assessment. In order to examine the clinical utility of FDG PET-CT, we analysed the data to determine the incremental change to TNM staging and alteration of distribution of lymph node level involvement, in addition to conventional staging. Abnormal areas of FDG avidity were defined as being above background, as previously described by Lonneux et al. . ChangesThe main limitation of this study is that pathological validation was not performed in any of these patients. This has also been the case in other series , 33. TheThe retrospective nature of this study is a further potential limitation. This is minimised by the interpretation at separate sittings of both imaging and radiotherapy plans by two radiologists and clinical oncologists, respectively. Although there is the possibility of selection bias of patients undergoing PET-CT, the TNM distribution of these stage III/IV patients is broadly similar to that in our previous report of the outcome of stage III/IV tonsil cancers . PET-CT 18FDG-PET has a higher sensitivity and specificity for lymph node involvement than CT or MRI. In the study by Lonneux et al. [In these 55 patients, TNM stage was altered by the FDG PET-CT findings in 17 31%) patients . This is% patientx et al. , FDG PETThe potential impact on clinical management is difficult to assess in a retrospective series and is inevitably subjective in nature. Only one patient in whom lung metastases were identified had a clear change in treatment intent/modality. In the 6 patients in whom FDG PET-CT downclassified nodal involvement, radiotherapy planning was felt to have been altered in 3 patients ; in one The impact of FDG PET-CT upon T-stage assessment has not been assessed in this study. Tumour size assessment based upon FDG PET is highly dependant upon the method of segmentation and no superiority over conventional imaging has been demonstrated .Refinements in imaging techniques are likely to lead to further improvements in the results obtained with functional imaging modalities. The results of FDG-PET-CT imaging are affected by differing acquisition protocols, image reconstruction, and interpretation techniques . StandarIn this series of patients with locally advanced HNSCC, FDG PET-CT altered the TNM stage and the identification of levels of nodal involvement in 31% and 40%, respectively. FDG PET-CT has potentially significant consequences for target volume definition in the IMRT era. The addition of imaging substudies to ongoing clinical trials will provide the ideal platform to optimise FDG PET-CT protocols and to further define a role in the management of HNSCC.


Three models for promoting male circumcision (MC) as a preventative intervention against HIV infection were compared among migrant worker populations in western China.A cohort study was performed after an initial cross-sectional survey among migrant workers in three provincial level districts with high HIV prevalence in western China. A total of 1,670 HIV seronegative male migrants were cluster-randomized into three intervention models, in which the dissemination of promotional materials and expert- and volunteer-led discussions are conducted in one, two, and three stage interventions. Changes in knowledge of MC, acceptability of MC, MC surgery uptake, and the costs of implementation were analyzed at 6-month and 9-month follow-up visits.All three models significantly increased the participants’ knowledge about MC. The three-stage model significantly increased the acceptability of MC among participants and led to greatest increase in MC uptake. At the end of follow-up, 9.2%  of participants underwent MC surgery; uptake among the one-, two-, and three-stage models were 4.9%, 9.3%, and 14.6%, respectively. Multivariable Cox regression analysis showed that three-stage model was the most effective method to scale up MC, with RR = 2.0  compared to the on-site session model. The two-stage intervention model showed no significant difference with either the on-site session model  or three-stage model (P=0.10).A three-stage intervention with gradual introduction of knowledge led to the significantly increase in MC uptake among migrant workers in western China, and was also the most cost-effective method among the three models. Epidemiological evidence indicates that the epidemic of HIV/AIDS in China has seen a distinct increase in heterosexual transmission in recent years –3. An esA systematic review and meta-analysis of randomized controlled trials in southern Africa has shown that MC reduced the risk of HIV infection by 38-66% over 24 months in heterosexual men –15. As wOur preliminary survey results showed that the rate of willingness to accept MC among male migrants in Guangxi, Xinjiang and Chongqing averaged 38.1% (manuscript in submission). Education about the benefits of MC for this population is therefore needed in western China. However, specific guidelines on conducting interventions among migrant workers have not yet been developed. Many routine HIV prevention programs in China currently include information on MC in one-time on-site education sessions during the annual Chinese New Year holidays, when many migrants return to their hometowns and are exposed to residency-based public health campaigns, but their effectiveness has not been systematically evaluated . To explThe study was part of a larger study which investigated the factors influencing Chinese males’ willingness to undergo circumcision . This stTo be eligible for our intervention study, blood samples of all participants were screened by ELISA (enzyme linked immunosorbent assay) and RPR (Rapid Plasma Reagent) to confirm sero-negative status for HIV and syphilis. Urethral or penile swabs were taken if urethral discharge or genital ulcers were available. We excluded previously circumcised men and those who could not answer the questionnaires independently.In total, 1,815 male migrant workers were invited to participate. Of these, 145 people were excluded, consisting of 135 who refused to be followed up, 5 who were HIV sero-positive, and 5 who were already circumcised. The resulting 1,670 male migrants enrolled were allocated to three different MC-related education intervention groups, through cluster randomization by work site. All subjects at the same work site received the same intervention. MC surgery was available free of charge at the local hospitals , where surgeons received MC-specific training and qualification assessments from the study researchers, and provided free postoperative treatment and consultation .We obtained written informed consent from each participant prior to their enrollment. Approval for this study, including study protocol and the circumcision program, was obtained from the Ethics and Human Subjects Committee of Guangxi Medical University.The content of the intervention models included a series of general and special promotional media materials. The general printed texts are brochures and orientation panels named the “Handbook of MC as Protection Against AIDS Transmission”, which includes the scientific context of MC, MC and AIDS prevention, benefits of MC in reproductive and sexual health, do’s and don’ts during the perioperative period, and so on. The general audio-visual productions consist of flash animation and scenario educational films about how MC protects people from HIV infection. The special printed texts also consist of both brochures and orientation panels but are different in content, which presented knowledge on MC through experiences of medical university students who underwent MC, especially the psychological process of learning about and accepting MC. The special audio-visual productions had the same content as the special printed texts. In addition, the special promotional program included on-site sessions and discussions led by invited experts and university student volunteers.Three intervention models were compared . The staP < 0.05. In addition, cost-effectiveness analysis was conducted for each model by calculating the cost of implementation  for every subject who underwent MC surgery.All data were double entered into EpiData software  and analyzed using SPSS for windows Version 19.0 . Descriptive statistics was generated for each variable corresponding to specific questions in the survey, including general characteristics and reasons for willingness or unwillingness to accept MC. Cost effectiveness analysis was performed on the total cost of each intervention model averaged over the number of MC surgeries undertaken by participants within the model. Cox-regression analysis was conducted to compare different types of interventions, with adjusting for demographic and migration characteristics and estimated relative risk (RR) with 95% confidence intervals. The baseline time was defined as the start point. The end point was either MC surgery uptake or the end of the second follow-up session, whichever came first. The incidences of HIV and syphilis infection in circumcised and non-circumcised subjects were compared using chi-squared test. All statistical tests were two-sided with significance level of A total of 1,670 male migrants were cluster-randomized by work site into three intervention groups: on-site session model (n = 487), two-stage intervention model (n = 547), and three-stage model (n = 636). At the sixth month after intervention, 460 (72.3%), 513 (93.8%), and 430 (88.3%) participants were followed up for each group respectively. At the ninth month of follow-up, 1,021 provided valid responses to the final questionnaire. The three models had no significant differences in self-reported religious affiliations, drug abuse history, and average scores of general knowledge about AIDS. There were statistically significant differences between the three models in terms of age, ethnicity, marital status, education level, place of origin, sexual history, and select STIs statuses, although their distribution showed generally consistent trends across the three models .The models were compared for their effectiveness in changing participants’ knowledge, attitudes and practices (KAP) regarding MC in follow-up sessions at the sixth month and ninth month after the interventions’ completion. Results showed that all three models led to statistically significant increases in KAP scores at both follow-up times compared to baseline, for almost every category and item of the assessment . The twoWe assessed the participants’ reported willingness to accept MC as well as actual MC uptake following the interventions. Uptake of MC surgery was statistically different among the three intervention models, with the three-stage model being the most effective . A totalRR= 2.0, 95% CI, 1.3-3.1, P=0.01). The two-stage intervention model showed no significant effect when compared with either the on-site session model  or the three-stage model (P=0.10).Cox proportional relative risk (RR) regression model was performed to compare the effects of different intervention models on the uptake of MC in migrant workers . ResultsThe costs and cost-effectiveness of the intervention models were compared. The input cost was defined as the total cost of the model divided by the number of recipients who received the model, while the cost effectiveness was assessed by dividing the total cost with the number of participants who uptook MC surgery due to the model’s interventions. The total costs included all expenses for developing the promotional materials, performing services, training, travel, and on site investigation and intervention. The input cost for the on-site session model was 45.3 RMB ($7.11 USD); for the two-stage model, 74.4 RMB ($11.68 USD); and for the three-stage model, 109.8 RMB ($17.24 USD). On the other hand, the cost per successful MC uptake for the on-site session model was highest at 929 RMB ($145.76) per case; the two-stage intervention model cost 798 RMB ($125.20) per case; and the three-stage model, 753 RMB ($118.14) per case. The cost per successful uptake showed that the three-stage model was significantly more cost effective than either the two-stage or the on-site session model. One-way sensitivity analysis for the cost per MC uptake showed that varying the most significant cost contributor by up to 10% did not change the nature of the cost effective comparisons: the three-stage model remained significantly more cost effective than either the two-stage or the on-site session model.The rates of HIV and syphilis infections acquired during the follow-up period were also compared between subjects who underwent MC and those who did not. HIV and syphilis incidences were zero among those who had undergone MC, while those who did not undergo MC had one case (incidence: 0.21 per 100 person-years) for both diseases.Circumcision has been shown in RCTs to be one of the most effective ways to reduce men’s risk of HIV infection through heterosexual contact –15. CompOur study resulted in an overall MC prevalence of 9.2% among participants, equivalent to 16 MC operations per month, and a prevalence of 14.6% among those who received the third intervention model. These are low compared with sub-Saharan Africa, where the median proportion of uncircumcised men willing to become circumcised was 65% (range 29–87%) across nine countries and thirteen studies . HoweverOur results indicated that a multi-staged intervention that gradually reinforces knowledge from promotional materials to peer and educator led discussions is the most effective model to promote MC in China. The involvement of peers, partners, and medical college students likely played a key role in encouraging participants to retain and internalize knowledge on MC and to take action on seeking MC surgery. As well, while the three intervention models did not differ in their ability in changing the participants’ willingness towards MC, the three-stage model was significantly more effective in promoting MC surgery uptake. This suggests that self-reported attitude towards MC is not strongly correlated with actual behavior in this population.Our study is subject to some limitations. We conducted two follow-ups after baseline, at six months  and at nine months . The second time point saw a high loss to follow up in Xinjiang, where severe winter weather conditions and social unrest led to low response rates, while Guangxi and Chongqing had 79% follow up. As well, while the prevalence of HIV and syphilis among uncircumcised men was higher than in circumcised men, this was not statistically significant. This may be due to a small sample size or an insufficiently long observation time. Nonetheless, our results demonstrate that a promotion model for MC consisting of three stages and progressively interactive interventions can increase circumcision among immigrant workers in western China. To explore the effectiveness of MC on reducing sexual transmission of HIV among migrant workers, future studies having an expanded sample size, longer postoperative observation time, and improved quality of pre- and post-operative counseling are necessary.


Whole-exome sequencing has identified the causes of several Mendelian diseases by analyzing multiple unrelated cases, but it is more challenging to resolve the cause of extremely rare and suspected Mendelian diseases from individual families. We identified a family quartet with two children, both affected with a previously unreported disease, characterized by progressive muscular weakness and cardiomyopathy, with normal intelligence. During the course of the study, we identified one additional unrelated patient with a comparable phenotype.We performed whole-genome sequencing (Complete Genomics platform), whole-exome sequencing , SNP genotyping (Illumina HumanHap550 SNP array) and Sanger sequencing on blood samples, as well as RNA-Seq (Illumina HiSeq platform) on transformed lymphoblastoid cell lines.From whole-genome sequence data, we identified RBCK1, a gene encoding an E3 ubiquitin-protein ligase, as the most likely candidate gene, with two protein-truncating mutations in probands in the first family. However, exome data failed to nominate RBCK1 as a candidate gene, due to poor regional coverage. Sanger sequencing identified a private homozygous splice variant in RBCK1 in the proband in the second family, yet SNP genotyping revealed a 1.2Mb copy-neutral region of homozygosity covering RBCK1. RNA-Seq confirmed aberrant splicing of RBCK1 transcripts, resulting in truncated protein products.While the exact mechanism by which these mutations cause disease is unknown, our study represents an example of how the combined use of whole-genome DNA and RNA sequencing can identify a disease-predisposing gene for a novel and extremely rare Mendelian disease. Over 5,500 confirmed Mendelian diseases have been described in the Online Mendelian Inheritance in Man (OMIM) database as of June 2013, but a third of them do not have a known molecular basis. With the rapid development and deployment of next-generation sequencing techniques, this situation is changing rapidly . HoweverThe current study was initiated by the discovery of a potential novel syndrome at The Children's Hospital of Philadelphia in 2009. We were presented with a brother and sister with a previously uncharacterized condition, which was initially suspected to be a glycogen storage disease type IV. Both children had progressive muscular weakness (myopathy) and cardiomyopathy, with normal intelligence Table , and theIn 2009, the two affected siblings and their parents were recruited to participate in a study to identify disease genes Figure . The proAll genome-wide SNP genotyping for the family was performed using the Illumina HumanHap550 BeadChip at the Center for Applied Genomics at the Children's Hospital of Philadelphia. Standard data normalization procedures and canonical genotype clustering files provided by Illumina were used to process the genotyping signals and generate genotype calls.The Illumina GenomeStudio software was used to process genotyping data and visualize signal intensity patterns at large-scale copy number variants (CNVs) and region-of-homozygosity (ROH) events. The log R ratio and B allele frequency measures for all markers for all samples were directly calculated and exported from the Illumina BeadStudio software. The CNV calls and ROH calls were generated using PennCNV (version 2009Aug27) , which u10[P/P], representing the confidence in the call. We removed variants that do not pass the default quality filter, including homozygous calls with quality scores less than 20, or heterozygous calls with quality scores less than 40. The variants passing the quality control threshold were used for downstream analysis.The whole-genome sequencing was performed by Complete Genomics , and we provided 10 μg DNA samples to the company for the sequencing service. The DNA was sequenced with a nanoarray-based short-read sequencing-by-ligation technology , includiThe whole-exome sequencing was performed in house at the University of Pennsylvania. We used the Agilent SureSelect Human All Exon kit for exon capture on 5 μg input DNA samples, and then used the Illumina Genome Analyzer II platform for next-generation sequencing. We generated 137 million paired-end reads, using two separate lanes from the Genome Analyzer. Data analysis was performed using the SeqMule pipeline , which iSelected putative variants were examined among all family members using Sanger sequencing methods. Given the position of variants, the PCR primers were designed to encompass the candidate position, ensuring that common SNPs are not covered by the primers. The ABI Prism 3500 sequencer was used for sequencing, and the resulting *.AB1 files were loaded into the ABI Sequence Scanner version 1.0 for further analysis and genotype calling. All sequence traces were manually reviewed to ensure the reliability of the genotype calls.We used the ANNOVAR software  for variWe generated Epstein-Barr virus-transformed lymphoblastoid cells for all study participants, using their peripheral blood mononuclear cells. Total RNA was extracted from cultured cells, and we made sequencing libraries using the Illumina TruSeq protocol. The Illumina HiSeq2000 sequencer was used for generating paired-end sequence data with 101 bp read length. We used the Tophat  version de novo CNVs or homozygous deletions shared by the two siblings. Therefore, in 2010, we started to use next-generation sequencing to comprehensively assay the genome of the patients, motivated by the successful identification of disease-predisposing genes for Mendelian diseases such as Miller syndrome and Kabuki syndrome [SNP genotyping revealed multiple CNVs in the two probands in the first family Figure . Howeversyndrome ,25 publiAt the time of the study, whole-genome sequencing was prohibitively expensive, and the relative merits of whole-genome versus whole-exome sequencing were not well established. Therefore, we decided to proceed with a modified approach, by sequencing one patient with whole-genome sequencing and the other by whole-exome sequencing. The whole-genome sequencing was performed by Complete Genomics, and an average fold coverage of 81× was achieved genome-wide with excellent evenness, ensuring high quality genotype calls for the patient. We identified 3,910,156 genetic variants for subsequent functional annotation and prioritization. In parallel, we performed exome sequencing in-house on the other patient. We generated 137 million paired-end reads, achieving an average coverage of 118× over designed capture regions, and with >90% of target regions covered by ≥10 reads. Therefore, the whole-genome and whole-exome data have excellent coverage statistics, even by today's standards.KCNJ12, HYDIN), olfactory receptors , as well as 'dispensable' genes with high frequency loss-of-function mutations in populations [LRP5 and MUC6. Additionally, while we were able to validate the mutations in FAM81B and MTRNR2L1, the mutations do not follow expected inheritance patterns (both mutations come from the same parent). Therefore, two genes were left as our final set of candidate genes, including TATA box-binding protein-associated factor 1-like  and RanBP-type and C3HC4-type zinc finger containing 1 .We first analyzed the whole-genome data to find potential disease-predisposing genes, assuming a recessive disease model as the most likely possibility, given that this is a brother and sister pair arising from phenotypically normal parents. We utilized the ANNOVAR 'variants reduction' pipeline  to identulations ,26, and TAF1L is a single-exon gene that evidently arose by retrotransposition of a processed TAF(II)250 mRNA during primate evolution [volution . TAF1L ivolution . The twoRBCK1 is an E3 ubiquitin-protein ligase that has a pivotal role in determining the specificity of the system by recognizing target substrates [RBCK1 is a more promising disease candidate gene, as it has two truncating mutations each inherited from one parent. These include a nonsense mutation in exon6 (p.Q222X) and a 7 bp frameshift insertion in exon5 (p.E190fs)  have been implicated in multiple Mendelian diseases sharing similar phenotypic features as the current patients.bstrates . RBCK1 i) Figure . We noteRBCK1 did not confirm as a candidate gene in the whole-exome data. The genome-wide variance of the heterozygous allele frequencies [RBCK1 were also well covered  using Illumina HiSeq2000. On average, we generated 61 million 101 bp paired-end reads for each subject.RBCK1 gene, originally found by Sanger sequencing , RNA-Seq data convincingly demonstrated the presence of a homozygous mutation at the splicing donor site in exon 5 of the RBCK1 was not well characterized, but it was reported to be a component of E3 ubiquitin-protein ligase, which accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes, such as UBE2L3/UBCM4, and then transfers it to substrates [HOIL1 (RBCK1) deficiency to a fatal human disorder with immunodeficiency, autoinflammation and amylopectinosis [RBCK1 gene may lead to distinct and unrelated phenotypes (immune-related problems and amylopectinosis). Despite the lack of direct functional evidence associating the mutations with the amylopectinosis phenotype, the discovery of a genetic cause further establishes that this phenotype of interest may represent a novel syndrome.The gene function for bstrates . Recentlctinosis . The autRBCK1 as possibly predisposing to a novel, extremely rare Mendelian disease. Together with several recently published studies [In conclusion, whole-genome sequencing identified a mutation in  studies , this exbp: base pair; CNV: copy number variant; IL: interleukin; NCBI: National Center for Biotechnology Information; PCR: polymerase chain reaction; ROH: region of homozygosity; SNP: single nucleotide polymorphism.The study was partly supported by a donation from the first family described in the article.KW: RG, YG, JB and GL analyzed the data and advised on data analysis. FGO and CH generated PCR and Sanger sequencing results. KT and CK generated the SNP genotyping results. ET helped with phenotype assessment. KW drafted the manuscript, and all authors reviewed and edited the manuscript. KW and HH designed the study and HH supervised all aspects of the study. All authors read and approved the final manuscript.Figures S1, S2 and S3.Click here for file


Targeted genomic enrichment (TGE) is a widely used method for isolating and enriching specific genomic regions prior to massively parallel sequencing. To make effective use of sequencer output, barcoding and sample pooling (multiplexing) after TGE and prior to sequencing (post-capture multiplexing) has become routine. While previous reports have indicated that multiplexing prior to capture (pre-capture multiplexing) is feasible, no thorough examination of the effect of this method has been completed on a large number of samples. Here we compare standard post-capture TGE to two levels of pre-capture multiplexing: 12 or 16 samples per pool. We evaluated these methods using standard TGE metrics and determined the ability to identify several classes of genetic mutations in three sets of 96 samples, including 48 controls. Our overall goal was to maximize cost reduction and minimize experimental time while maintaining a high percentage of reads on target and a high depth of coverage at thresholds required for variant detection.We adapted the standard post-capture TGE method for pre-capture TGE with several protocol modifications, including redesign of blocking oligonucleotides and optimization of enzymatic and amplification steps. Pre-capture multiplexing reduced costs for TGE by at least 38% and significantly reduced hands-on time during the TGE protocol. We found that pre-capture multiplexing reduced capture efficiency by 23 or 31% for pre-capture pools of 12 and 16, respectively. However efficiency losses at this step can be compensated by reducing the number of simultaneously sequenced samples. Pre-capture multiplexing and post-capture TGE performed similarly with respect to variant detection of positive control mutations. In addition, we detected no instances of sample switching due to aberrant barcode identification.Pre-capture multiplexing improves efficiency of TGE experiments with respect to hands-on time and reagent use compared to standard post-capture TGE. A decrease in capture efficiency is observed when using pre-capture multiplexing; however, it does not negatively impact variant detection and can be accommodated by the experimental design. Massively parallel sequencing has expanded the genomics era by dramatically reducing the cost and time of large-scale DNA sequencing. Although whole genome sequencing may soon become routine, in terms of cost, time, and labor, it is often more practical to target specific regions of interest in the genome. Targeted genomic enrichment (TGE), also known as targeted sequence capture, allows efficient isolation of genomic regions prior to massively parallel sequencing . BrieflyTGE has been used to discover mutations in sets of genes associated with specific diseases -5 or, inThe increase in sequencer output has well outpaced our ability to efficiently use the sequence generated. While it is clear that saturating levels of sequencing coverage are required for the lowest false positive and false negative rates in TGE experiments , it is aWhen applied to TGE, multiplexing can be performed prior to capture (pre-capture multiplexing) or after capture (post-capture multiplexing). The first protocols using molecular barcodes employed a post-capture approach  and postHowever, the limits of pre-capture multiplexing have not been thoroughly tested. Of two pilot studies showing the feasibility of pre-capture multiplexing one used 8 samples and single pools of 3 or 5 samples pooled pre-capture , while aIn general, post-capture multiplexing is used for TGE whereas pre-capture multiplexing is not. In this study, we sought to address several questions germane to multiplexing during TGE experiments, including the effect of inter-sample competition for capture baits during hybridization, the impact on capture efficiency, and the downstream effects on overall sequence quality as measured by read mapping and duplicate reads. To address these questions, we compared standard post-capture multiplexing to pre-capture multiplexing using two pool sizes (n=12 or 16 samples per pool) to study a large set of samples (n = 96). We demonstrate the significant advantages of pre-capture multiplexing in cost and time reductions while at the same time maintaining our minimum threshold for accurate variant detection.We compared standard post-capture multiplexing with pre-capture multiplexing for TGE of a relatively small genomic region of 521,647 bp that comprises all known non-syndromic deafness and Usher syndrome genes as described previously . We perfT-test p < 0.01 for both pre-capture 12 and 16), however in all cases, greater than 90% of reads mapped, constituting a high-quality sequencing run in our experience.The total number of sequencing reads was significantly lower in pre-capture lanes as compared with the post-capture lane , however, the two pre-capture lanes were not significantly different. 97.4%, 93.3%, and 91.6% of sequencing reads mapped on average for post-capture, pre-capture 12, and pre-capture 16, respectively Figure . The perWe define capture efficiency as the percent of all mapped reads that overlap the targeted regions. In our experience using SureSelect TGE, capture efficiency ranges from ~40% for small target regions (<200kb) to up to ~80% for large target regions  when using standard post-capture multiplexing (data not shown). On average, the capture efficiency for post-capture samples was 68.7% Figure . This waDepth of coverage represents the number of sequencing reads aligned over a sequenced base pair. On average, all methods of multiplexing showed a 1X coverage Figure  that wasWe found a difference in coverage distribution among multiplexing methods Figure . The disIn order to obtain a similar depth of coverage as post-capture samples when performing pre-capture 16 multiplexing, fewer pools of 16 can be sequenced in a single lane as a compensation for reduced capture efficiency. To test this hypothesis, we randomly sub-sampled sequencing reads from the pre-capture 16 lane to simulate a total of 5, 4, and 3 pools of 16 samples  per lane Figure  and B. WWe used 96 unique DNA samples in three sets of 96 captures each. In each set of samples, 48 samples were from persons with presumed genetic hearing loss of an unknown cause and the results from these samples will be reported elsewhere. 48 samples in each set were either positive controls carrying deafness-causing mutations verified by Sanger sequencing or negative controls . 100% of variants were identified in all three lanes using our variant calling and annotation pipeline (see Methods) with the exception of the sample from post-capture lane that failed at sequencing , as this variant was found in all three lanes and confirmed with Sanger sequencing.We performed a systematic analysis to examine the possibility of artifacts or sequencing errors associated with molecular barcoding that may lead to erroneous sample assignment (chimeras) and misdiagnosis. We searched within each lane for positive control variants present in the un-annotated (“raw”) variant call format (VCF) files for any of the samples present in any other sample in that lane even at low observation rates. We did not find any evidence of sample-switching due to aberrant barcode identification in any lanes, although we did find that a single individual was an incidental carrier for a disease-causing mutation in the th or 1/12th of the cost of post-capture TGE. As an example, each SPRI-bead purification during the protocol costs approximately $5.76/purification. Using pre-capture 16 multiplexing, samples are pooled for three of these purifications compared to post-capture TGE. For 96 samples, the cost simply for purification of each of these samples individually three times  is $1,658.88. Pre-capture 16 multiplexing would reduce the number of purifications required to 6 samples three times (18 purifications) and therefore the cost is $103.68 or a cost reduction of 93.75%. The same calculation yields a cost of $138.24 for pre-capture 12 multiplexing and a corresponding cost reduction of 91.7%. Similar reductions in cost can be assumed for other reagents and consumables.Pre-capture multiplexing was associated with reduced capture efficiency, which in turn reduced average depth of coverage but did not negatively impact variant detection. Significant savings in cost and time were associated with pre-capture multiplexing. Costs for XTv2 kits are 15% lower than XTv1. There are other costs that are associated with library preparation including consumables which are difficult to quantify, but can be estimated as 1/16In our hands, TGE requires 6 hours for pre-hybridization steps and 4 hours for post-hybridization steps. Post-capture multiplexing introduces barcodes at the final amplification step and pooling is completed immediately prior to sequencing, which does not reduce hands-on time. Pre-capture multiplexing occurs prior to the hybridization and therefore 12X or 16X as many samples can be hybridized and captured in the same amount of time. Therefore, though difficult to quantify, hands-on time is significantly reduced with pre-capture multiplexing.Massively parallel sequencing and TGE enable rapid and efficient sequencing of hundreds of thousands or millions of base pairs of the human genome simultaneously. While prices have decreased drastically, efficiency and cost-effectiveness are still important considerations, especially when large numbers of samples are analyzed. In this study we provide the first systematic comparison of post- and pre-capture multiplexing on a large sample set.We used a large number of positive control mutations to validate this capture method, and in the process uncovered a variety of genomic variants including small and large deletions, and single nucleotide variants in control patients. We found no evidence of barcode switching or erroneous barcode assignment. In addition, the allelic balance, which is important for making heterozygous calls, was not affected by pre-capture pooling.As expected, our data show that multiplexing samples prior to hybridization and capture reduces capture efficiency. Importantly, we noted a decrease in duplicate reads, which may partially offset this loss in efficiency. We used simulations to show that reduced efficiency can be compensated for by modifying the experimental design . We hypothesize that this effect is due to competition for complementary RNA baits among multiple genomes, specifically because the effect was more pronounced with the higher pool size (pre-capture 16). In addition, we found a difference in coverage distribution when comparing multiplexing methods Figure , with thCosts are an important factor in any experimental design. Kit costs for pre-capture multiplexing are 15% lower. However, greater cost savings are found in the reduction in consumables and ancillary reagents used when pre-capture multiplexing is employed. In our example, we show that costs of purifications alone are reduced by ~94% for pre-capture 16 multiplexing and ~92% for pre-capture 12 multiplexing. The most significant reduction when using pre-capture multiplexing lies in reduced hands-on time. Although this is difficult to quantify, 12X or 16X as many samples can be processed after pooling occurs when pre-capture multiplexing is used. Therefore, due to the greatest reduction in costs and hands-on time, as well as a lack of detrimental effects on quality of TGE, pre-capture 16 represents the most effective and efficient method for TGE of an experiment with a similar target size as shown here . When designing their own experiments, investigators can estimate the optimum number of samples to pool pre-capture and sequence per lane based on the effects on capture efficiency described here.Here we show for the first time the effect of pre-capture multiplexing during TGE on a large set of samples. We noted a specific effect on capture efficiency during pre-capture multiplexing and we hypothesize that this effect is due to multiple genomes competing for hybridization with complementary RNA. However, pre-capture multiplexing provided significant cost-savings and time reductions, resulted in no barcode mis-identification, and could reliably identify several classes of genetic variation. In summary, pre-capture multiplexing increases the efficiency of TGE and massively parallel sequencing to identify genomic variants.This study was approved by the University of Iowa Institutional Review Board. We used 96 samples including DNA from the following individuals: 39 positive controls, 9 negative controls, and 48 unknowns. The positive control individuals had non-syndromic hearing loss (NSHL) and in total represented 44 mutations including small indels, large deletions and missense mutations previously diagnosed with Sanger sequencing ; and local realignment and variant identification was completed with GATK [Bioinformatics analysis was performed using a local installation of the Galaxy software and a custom analysis pipeline, as previously described  with modith GATK . We usedith GATK , Bedtoolith GATK  and NGSRith GATK . Copy nuith GATK .TGE: Targeted genomic enrichment; MPS: Massively parallel sequencing.SJ, HR, ACG, BN, SH, and EML are employees of Agilent Technologies Inc., which manufactures and offers for commercial sale targeted genomic enrichment kits like the one used in this work. AES, MSH, and RJHS have no competing financial interests.AES, MSH, RJHS, SH, and EML designed the experiment. AES, MSH, HR, and SJ performed the experiments. BN designed the sequence capture baits. AES, SJ, HR, ACG, and RJHS analyzed the data. AES, MSH, and RJHS drafted the manuscript. All authors read and approved the final manuscript.Table S1. Positive Control Mutations.Click here for fileTable S2. 8 bp barcodes used.Click here for file


Several EGFR-tyrosine kinase inhibitors (EGFR-TKIs) including erlotinib, gefitinib, afatinib and icotinib are currently available as treatment for patients with advanced non-small-cell lung cancer (NSCLC) who harbor EGFR mutations. However, no head to head trials between these TKIs in mutated populations have been reported, which provides room for indirect and integrated comparisons.We searched electronic databases for eligible literatures. Pooled data on objective response rate (ORR), progression free survival (PFS), overall survival (OS) were calculated. Appropriate networks for different outcomes were established to incorporate all evidences. Multiple-treatments comparisons (MTCs) based on Bayesian network integrated the efficacy and specific toxicities of all included treatments.P<0.00001; 1-year PFS: 42.9% vs. 9.7%, OR 7.83, 95%CI 4.50 to 13.61; P<0.00001) through direct meta-analysis. In the network meta-analyses, no statistically significant differences in efficacy were found between these four TKIs with respect to all outcome measures. Trend analyses of rank probabilities revealed that the cumulative probabilities of being the most efficacious treatments were : erlotinib , gefitinib , afatinib  and icotinib , respectively. However, afatinib and erlotinib showed significant severer rash and diarrhea compared with gefitinib and icotinib.Twelve phase III RCTs that investigated EGFR-TKIs involving 1821 participants with EGFR mutation were included. For mutant patients, the weighted pooled ORR and 1-year PFS of EGFR-TKIs were significant superior to that of standard chemotherapy (ORR: 66.6% vs. 30.9%, OR 5.46, 95%CI 3.59 to 8.30, The current study indicated that erlotinib, gefitinib, afatinib and icotinib shared equivalent efficacy but presented different efficacy-toxicity pattern for EGFR-mutated patients. Erlotinib and afatinib revealed potentially better efficacy but significant higher toxicities compared with gefitinib and icotinib. Lung cancer is the leading cause of cancer-related mortality worldwide, with about 85% patients suffering from non-small cell lung cancer (NSCLC) Network meta-analysis, also known as multiple-treatments comparison, enables us to synthesize data from both direct  and indirect comparisons  of diverse regimens We searched PubMed, EMBASE and the Central Registry of Controlled Trials of the Cochrane Library using the combination of the search terms “non-small cell lung cancer”, “epidermal growth factor” OR EGFR, AND mutation within the restriction limit of “randomized controlled trial” (the deadline was March 2013). To identify updated outcomes from included trials or unpublished trials that had presented analyzed data, we also reviewed abstract books and presentations of major recent meetings of American Society of Clinical Oncology (ASCO), European Society for Medical Oncology (ESMO) and World Conference on Lung Cancer in 2008–2012. Finally, the reference lists of the included studies were reviewed as a supplement. No language limits were applied.The eligible studies should be phase III RCTs that compared one TKI  to another or to standard chemotherapy as first-line or second-line treatments in patients with advanced NSCLC that presents activating EGFR mutations. Since the dominant histological type of patients with EGFR mutation was nonsquamous carcinoma in which pemetrexed were proved to yield superior efficacy compared with other third-generation chemotherapy agents, we also included studies that compared pemetrexed-based regimen with pemetrexed-free regimen in order to optimize the network. Notably, advanced NSCLC was defined as stage III or IV disease that was not feasible to surgical treatment or radiotherapy. Phase III RCTs were defined as studies with a power greater than 0.80 to detect a difference in survival. EGFR mutations mainly referred to deletions in exon 19 or the L858R substitution in exon 21. Standard chemotherapy was defined as platinum-based third generation doublets for first-line treatments or pemetrxed/doctaxel for second-line treatments. In cases of overlap reports, we included only the latest results. Studies failed to meet the inclusion criteria will be excluded.http://www.cochrane.de). The data on major clinical features, overall survival (OS), progression free survival (PFS), objective response rate (ORR) and adverse events  were extracted by two investigators (LW and WX) independently. Figures were electronically digitized and Kaplan-Meier curves were downloaded by appropriate software . We rated the quality of each eligible study with JADAD score The data collection and assessment of methodological quality follows the QUORUM and the Cochrane Collaboration guidelines (2). Statistical significance was considered at P<0.05. All calculations were performed using REVIEW MANAGER .First, we conducted pair-wise meta-analyses with a random-effects model to synthesize studies comparing the same pair of treatments. The results were reported as pooled ORs with the corresponding 95% confidence interval (CI). Statistical heterogeneity across studies was assessed with a forest plot and the inconsistency statistic  We also estimated the probability that each of the treatment was the best regimen, the second best, the third best, and so on, by calculating the OR for each drug compared with an arbitrary common control group, and counting the proportion of iterations of the Markov chain in which each drug had the highest OR, the second highest, and so on. We ranked treatments in terms of efficacy and acceptability with the same methods.A variance calculation and a node-splitting analysis provided by the software ADDIS 1.15 were applied to evaluate the inconsistency within the network meta-analysis. If the difference between random effects variance and inconsistency variance was large or a P<0.05 of disagreement between direct and indirect evidence was met, significant inconsistency was indicated. According to the quantitative estimation, we could adjust the study inclusion and ultimately obtain an ideal network with consistency.We identified 1572 records according to the search strategy and finally included 12 phase III RCTs that compared elotinib, gefitinib, icotinib, afatinib or chemotherapy in chemo-naïve or previously treated advanced NSCLC patients P<0.00001). In terms of disease control, TKIs yielded 42.9% 1-year PFS  which was higher than that of chemotherapy 9.7%  with an OR of 7.83 . Since OS data of ICOGEN for mutation population was unavailable, OS were not calculated for icotinib. The pooled 1-year and 2-year OS of TKIs was 79.2%  and 49.7%  respectively. On the other hand, the OS outcomes for chemotherapy were 78.9%  and 51.0%  for 2-year. Inconsistent with the results of ORR and PFS, OS data were similar between TKIs and chemotherapy . For mutated patients, the weighted pooled ORR and PFS of EGFR-TKIs were significant higher than standard chemotherapy. The pooled ORR was 66.6%  for TKIs versus 30.9%  for chemotherapy with an OR of 5.46 , the results were : erlotinib , gefitinib , afatinib , whereas outcomes of icotinib were not assessable , it is difficult to integrate information on the relative efficacy of all tested regimens for the same indication. Similarly, conventional direct meta-analysis also fails to measure the relative effect between diverse treatments as it only synthesizes trials with a same pair of comparators. Multiple-treatments comparison (MTC), or so called network meta-analysis, could compare a set of treatments for a specific disease simultaneously through a common comparator treatment http://clinicaltrials.gov). Thus, no head-to-head comparison between these agents in EGFR-mutated populations has been available by far. These provided room and need for indirect and integrated comparisons. A recent pooled analysis of available studies was performed to evaluate clinical outcome in patients with EGFR-mutated NSCLC This network meta-analysis was the first study using appropriate statistical methods to provide indirect comparison for the currently available EGFR-TKIs  in treating patients with advanced NSCLC who harbor EGFR mutations, based on all available information from phase III randomized trials. The superior efficacy of EGFR-TKIs for mutated population compared to chemotherapy has been substantially proved For indirect methods, the underlying assumptions of the exchangeability of studies across the entire network should be examined carefully. We included only large phase III randomized trials with strict patient allocation and optimized balance between treatment and control arm to ensure the cross-study exchangeability. Besides, EGFR mutation status had been determined as the most remarkable predictor for EGFR-TKIs. In the current study, only patients with EGFR-mutation were included, which guarantee the homogeneity of study population. In addition, treatment-line might affect the efficacy and survival outcome of TKIs since a recent study suggested that chemotherapy might reduce EGFR mutation frequency Firstly, we generated a set of pooled data according to the weight of each study which illustrated the current status of treatment with EGFR-TKIs. Based on such data, we could more intuitively show the true benefits which were given rise by EGFR-TKIs compared with traditional chemotherapy no matter in within-trials comparison or historical contrast, rather than merely reporting the OR/HR value. The superiority of EGFR-TKIs in ORR and PFS for EGFR-mutated patients indicated its specific efficacy in suppressing the tumor cells that were driven by the EGFR pathway. The failure to make a distinction between the OS outcomes of TKIs and chemotherapy could be explained by the influence of subsequent treatments Based on both network 1 and network 2, it was manifested that all currently available EGFR-TKIs were comparable in terms of ORR, PFS and OS  as treatments for EGFR mutated NSCLC patients. In a population perspective, no statistical differences between agents were observed. Rank probabilities provide us another perspective to review the position of certain treatment among all, especially when the relative values fail to reach statistical significance. The ranking could tell us which treatment would most likely be the best option, or whether one treatment is potentially better than another. In the current study, erlotinib had the greatest probability to rank the first among all the four TKIs regarding ORR and 1-year PFS while afatinib rank best in 1-year and 2-year OS. Icotinib shared similar rankings with afatinib respect to ORR and 1-year PFS. Gefitinib was associated with relatively low probabilities to rank the first but showed similar ranking compared with erlotinib in 2-year OS.50 of erlotinib and icotinib for molecule level or cellular level was similar and significant smaller than that of gefitinib The trend of superiority of erlotinib versus gefitinib was in line with the previous pooled analysis Although presenting potentially better efficacy, erlotinib and afatinib were associated with significant higher toxicities compared with gefitinib and icotinib. Through reviewing the distribution of rank probabilities in This is the first multiple treatment comparison for the currently available EGFR-TKIs in treating EGFR mutated advanced NSCLC patients based on evidences with good quality. The finding that efficacy was equivalent among these agents might help clinicians in making decisions. However, there existed several limitations. First, OS data in mutant population of ICOGEN were not available hence we could not evaluate the survival benefits of icotinib. In addition, comparisons in terms of OS were confounded by subsequent treatments. Second, the performance of icotinib in first-line setting was not available. Third, we could not assess some important molecular markers including T790M status in the population which might have effects on the efficacy of TKIs and cause bias. Fourth, the established networks lacked sufficient direct comparisons between TKIs. At last, we could not evaluate the adverse events in mutant population in the current study since no specific data from these patients were reported by the included trials. As we know, the overall data of adverse events might be influenced by the proportion of mutated patients. Therefore, future head-to-head randomized trials which would optimize the network and multiple treatment comparison based on individual patient data are warranted to further clarify these issues. Novel TKIs in the second- or third- generation such as canertinib, dacomitinib and CO-1686 were expected to be included.In conclusion, this network meta-analysis indicated that erlotinib, gefitinib, afatinib and icotinib shared equivalent efficacy but presented different efficacy-toxicity pattern for EGFR-mutated patients according to current evidences. We suggested that physicians fully consider the efficacy-toxicity balance to select appropriate TKI for patients.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.File S1Table S1, Multiple treatment comparison for efficacy based on network 2 (1st-line studies only). Table S2, Rank probabilities of each TKI for different outcomes based on network 2 (1st-line studies only).(DOC)Click here for additional data file.


The relationship between HIV knowledge and HIV-related behaviors in settings like Mozambique has been limited by a lack of rigorously validated measures.A convenience sample of women seeking prenatal care at two clinics were administered an adapted, orally-administered, 27 item HIV-knowledge scale, the HK-27. Validation analyses were stratified by survey language (Portuguese and Echuabo). Kuder-Richardson (KR-20) coefficients estimated internal reliability. Construct validity was assessed with bivariate associations between HK-27 scores (% correct) and selected participant characteristics. The association between knowledge, self-reported HIV testing, and HIV infection were evaluated with multivariable logistic regression.Participants (N = 348) had a median age of 24; 188 spoke Portuguese, and 160 spoke Echuabo. Mean HK-27 scores were higher for Portuguese-speaking participants than Echuabo-speaking participants . Internal reliability was strong (KR-20>0.8) for scales in both languages. Higher HK-27 scores were significantly (p≤0.05) correlated with more education, more media items in the home, a history of HIV testing, and participant work outside of the home for women of both languages. HK-27 scores were independently associated with completion of HIV testing in multivariable analysis , but not with HIV infection.HK-27 is a reliable and valid measure of HIV knowledge among Portuguese and Echuabo-speaking Mozambican women. The HK-27 demonstrated significant knowledge deficits among women in the study, and higher scores were associated with higher HIV testing probability. Future studies should evaluate the role of the HK-27 in longitudinal studies and in other populations. In sub-Saharan Africa, there were an estimated 1.8 million persons newly infected with HIV in 2009, representing an 18% reduction in incidence since 2001 ad hoc questionnaire or interview to ascertain HIV knowledge and do not report the specific items or psychometric properties of the measure, limiting the reproducibility and comparability of findings Accurate knowledge about HIV/AIDS is an important, though insufficient, component of an individual's adoption of healthy behaviors ad hoc knowledge measures In Mozambique, the prevalence of HIV infection was 11.5% in 2009 The study protocol was reviewed and approved by the National Committee of Bioethics for Health in Mozambique  and the Institutional Review Board of Vanderbilt University. No financial incentive was provided to participants, and written informed consent was obtained for all study participants that included permission to review medical records relating to HIV testing.Zambézia Province is located in central Mozambique, is predominantly rural, and had an estimated adult HIV prevalence of 12.6% in 2009 The HK-27 was developed by adapting items from three existing HIV knowledge scales: the Behavioral Surveillance Survey (BSS) from FHI 360, Items measuring HIV knowledge from the HIV-KQ-45, BSS and DHSAIDS were pooled and reviewed by experts in HIV care for those living in resource-limited settings, cultural norms and beliefs surrounding HIV in Mozambique, health literacy, and survey design. Items were excluded if they were judged to be redundant in content, less culturally relevant for the Mozambican population, or poorly constructed. The resulting set of items assessed HIV knowledge across three content domains: general disease knowledge, sexual transmission risk factors, and non-sexual transmission risk factors. Four original items were created to add a fourth content domain, assessing knowledge of HIV treatment. A total of 30 items were included after preliminary review; these were translated into Portuguese by a fluent speaker and back-translated into English to verify the accuracy of the translation. The items were also translated by a trained bilingual translator from Portuguese into a local Mozambican language (Echuabo), a principal local language for southern Zambézia Province The two sets of translated survey items were assessed for clarity and cultural relevance with cognitive interviews of 32 women (19 Portuguese-speakers and 13 Echuabo-speakers) who were awaiting prenatal care at two Zambézia health centers, in Quelimane and Inhassunge. Interviews were conducted in Portuguese or Echuabo, and took place in the language of choice for each participant. A trained interpreter fluent in Portuguese and Echuabo was used to conduct cognitive interviews of Echaubo-speaking participants. These interviews assessed participants' understanding of each item, ensured translations were accurate, and verified all items included in the scale were culturally appropriate. As a result of the cognitive interviews, three items were discarded because of poor clarity, and the translation of 12 items underwent slight revision to better match local language usage, using the suggestions of interview participants. The final scale included 27 items; each item consisted of a statement for which a participant could respond “agree”, “disagree” or “uncertain”. As with the HIV-KQ-45, the response choice of “uncertain” was included to discourage guessing A convenience sample of participants was recruited during prenatal care at two health centers. Potential participants were approached after completing a prenatal care consultation. One site was urban (Quelimane) and served patients who commonly spoke Portuguese, a second site (Inhassunge) was rural and served patients who commonly spoke Echuabo; an effort was made to recruit participants proficient in each language at each site. Women were included if they were 1) pregnant, 2) ≥18 years of age, and 3) spoke either Portuguese or Echuabo as their primary language.After consent was obtained, women were interviewed in a private area of the hospital. All study measures were administered orally in the language of choice of the participant (Echuabo or Portuguese); a trained interpreter fluent in Portuguese and Echuabo was used to facilitate administration of the HK-27 to Echaubo-speaking participants. Sociodemographic characteristics, including age, number of children, education, and occupation, were ascertained by participant self-report. Each participant was administered the HK-27, after which any participant questions about content were addressed.Each HK-27 item was scored as correct or incorrect, with responses of uncertain counted as incorrect. HIV knowledge was defined as the percentage of HK-27 items answered correctly for each participant. Media item ownership was ascertained by self-reported ownership of television, internet, or cell phone and ranged from 0–3 items. HIV testing was ascertained by self-report to account for tests done at other clinical sites where medical records were inaccessible. HIV test results were obtained on review of the medical record at the study sites only. All data was recorded on paper and entered into a secure electronic database, REDCap™ Sociodemographic data were reported as medians with interquartile ranges (IQR) or as proportions where statistically appropriate for the total sample, and stratified by survey language. Mean HK-27 scores were tabulated along with standard deviations (SD) for the total sample, and separately by language. The proportions of women who obtained an HIV test and were HIV-infected were also tabulated.To ensure construct validity of the HK-27 in both languages, psychometric testing and validation of the Echuabo and Portuguese language scales were done separately. To establish construct validity of the HK-27, we hypothesized that higher HIV knowledge would be associated with characteristics that may indicate greater exposure to HIV-related information, such as older age, more children , receipt of HIV testing, and ownership of more media items. Other factors that may indicate a higher capacity for understanding HIV-related information  were also included in the assessment of construct validity. We hypothesized that Portuguese-speaking participants would have higher scores than participants who spoke Echuabo. Kuder-Richardson 20 coefficients were generated to test internal reliability of the HK-27. Spearman correlations were calculated to assess the association between HK-27 scores and continuous, nonparametric variables; Wilcoxon rank-sum tests compared HK-27 scores across binomial variables, and Kruskal-Wallis tests compared HK-27 scores for categorical variables.a priori based on the belief that these factors were related to health care access. All analyses were conducted using STATA™ statistical software package .Multivariable logistic regression models examined the relationship between HIV knowledge and self-reported HIV testing. A second set of logistic regression models tested the association between HIV knowledge and HIV-infection for those with medical records available to review. Multivariable models adjusted for study language and site, maternal work, and travel time to the hospital. Covariates in the multivariable model were chosen In June and July of 2011, 472 women completing prenatal care visits were approached at two clinics ; 348 gave consent to participate, yielding a response rate of 74%. Surveys were conducted in Portuguese (188 participants) and Echuabo (160 participants). The HK-27 took between 7–10 minutes for participants to complete. Women had a median age of 24 years (IQR: 20–28), had little formal education , and worked either inside the home or in agriculture (89%). Most (85%) reported they had obtained an HIV test in the past; 6% of women who reported HIV testing had been tested at another site. Of the 275 women who had undergone HIV testing at one of the study sites, 34% had HIV infection. Compared with the Portuguese speaking participants, Echuabo participants reported less education, had fewer participants report work outside the home, had more travel time from home to the clinic, reported more HIV testing, and had lower rates of HIV infection Table 1.Performance on the HK-27 was higher for Portuguese speakers  than for Echuabo speakers  (p<0.001). Internal reliability was strong for both scales: the Kuder-Richardson coefficient was 0.8 for the Portuguese language scale and 0.9 for the Echuabo language scale. Misconceptions about HIV transmission were very common, especially for Echuabo speaking participants, a majority of whom incorrectly responded HIV could be spread by shaking hands, sneezing, sharing utensils or by a curse. Many women of both languages incorrectly reported that cleaning the genitals after intercourse was protective against HIV transmission. Only half of Portuguese speaking women and 14% of Echuabo speaking women agreed that a person with HIV infection could otherwise look and feel healthy.Higher HK-27 scores were significantly correlated (p<0.05) with more education  and more media items in the home . Higher HK-27 scores were associated with fewer children at home for Portuguese speakers  but not significantly for Echuabo speakers. Higher HK-27 scores were significantly associated with work outside of the home as compared to domestic work/agriculture for women in both language groups. Women who reported obtaining a test for HIV had higher HK-27 scores than women who had not been tested . Women with HIV infection had lower HK-27 scores than those who had tested negative among Portuguese speakers (Mean HK-27 score: 66% vs. 71% p = 0.04); there were no statistically significant differences in HK-27 scores for Echuabo speaking women with positive vs. negative HIV test results Table 3.Mean HK-27 scores increased with higher educational attainment for both Portuguese and Echuabo speakers Table 4.In multivariable logistic regression modeling including data from all participants, HK-27 scores were significantly associated with reported HIV testing after adjusting for survey language, study site, travel time, and maternal work . There was no significant association in fully adjusted models between HK-27 scores and HIV infection for the 275 women with test results available to review in the medical record .The HK-27 is a valid and reliable measure of HIV knowledge for Portuguese and Echuabo speaking women seeking prenatal care in rural Mozambique, demonstrating strong internal reliability and meeting six of eight criteria selected to establish construct validity for the Portuguese instrument and five of eight criteria for the Echuabo instrument. Women in this study responded correctly to an average of 56% of items on the HK-27. Additionally, we found that HIV knowledge was independently associated with HIV testing, such that women that had higher levels of HIV knowledge had a higher likelihood of reporting that they had been screened for HIV in the past. By including relevant items from the BSS, This study adds important detail to what is known about the level of HIV knowledge gleaned from population-based public health surveys such as the DHS in high prevalence settings In this study, women who spoke Echuabo had lower levels of HIV knowledge than their Portuguese-speaking counterparts. Echuabo-speakers as a group had lower levels of education than Portuguese-speakers, possibly explaining the disparity in HIV knowledge between the groups. However, the observation that Echuabo-speakers had lower HIV knowledge compared to their Portuguese-speaking peers with similar levels of educational attainment would suggest that this is an incomplete explanation. Since Portuguese is the language commonly used in the clinical setting to counsel patients and is often also used in public health educational efforts in Mozambique, Women at both study sites reported a high degree of HIV screening relative to estimates for Zambézia Province from 2009 Other limitations include the cross-sectional design of the study, which limits the ability to define causality in our observed relationship between HIV knowledge and HIV testing. Our findings may not be generalizable to men or to women over childbearing age. Especially for Echuabo participants, our validation analyses were limited by the low level of participant education and limited variation of work type for women in our study. While we enrolled women at two sites, demographic characteristics of women living in Quelimane and Inhassunge were such that most of the Portuguese-speaking participants were enrolled in Quelimane, and most of the Echuabo-speaking participants lived in Inhassunge. While we adjusted for study site in multivariable models that tested associations between HIV knowledge and testing, it is possible that unmeasured confounders related to rural vs. urban settings influenced our findings. Especially in urban Quelimane, women had opportunities to obtain HIV screening in settings separate from where prenatal care was delivered, and the study was limited by the inability to review medical records at other clinical sites, obligating the use of self-reported HIV testing that introduces the possibility of bias. Since illiteracy is very common among women in Zambézia Province, Our study introduces an adapted instrument to measure HIV knowledge that is contextually relevant for use in sub-Saharan Africa, highlights some important gaps in knowledge in two sites in rural Mozambique, and demonstrates a potentially important relationship between knowledge and HIV testing. Our experience adapting the HK-27 into two languages, including a traditional language, suggests that the measure could be adapted into other languages to measure knowledge in other settings in sub-Saharan Africa. While the HK-27 was adapted to use in research efforts, there are other potential applications to the HK-27. First, the measure could be used to assess gaps in knowledge on a community or clinic-level to better tailor HIV-related public health efforts to the informational needs of a given population. Second, the measure could be used as an evaluation tool for HIV-related trainings of counselors, community activists, and health staff. Third, a shortened instrument could be given to individual women prior to their prenatal care counseling, and could be used to focus counseling messages to address gaps in knowledge and avoid message redundancy. Finally, although we found preliminary evidence to support the relationship between HIV knowledge and testing, prospective studies are needed to understand exactly how HIV knowledge impacts health behaviors in sub-Saharan Africa.