Vitamin D does not only regulate calcium homeostasis but also plays an important role as an immune modulator. It influences the immune system through the induction of immune shifts and regulatory cells resulting in immunologic tolerance. As such, vitamin D is thought to exert beneficial effects within the transplant setting, especially in kidney transplant recipients, considering the high prevalence of vitamin D deficiency in kidney transplant recipients.3) within the transplant setting. Kidney transplant recipients found to have vitamin D deficiency defined as 25-hydroxyvitamin D3 < 50 nmol per liter will be randomly assigned to receive either oral cholecalciferol therapy or placebo and will be followed for one year. Cholecalciferol will be administered at a dose of 6800 International Units daily over a time period of one year.The VITA-D study, a randomized, placebo-controlled, double-blind study with two parallel groups including a total of 200 kidney transplant recipients, is designed to investigate the immunomodulatory and renoprotective effects of cholecalciferol  one year after kidney transplantation will be evaluated. Incidence of acute rejection episodes, and the number and severity of infections  within the first year after transplantation will be monitored as well. As a secondary endpoint the influence of vitamin D3 on bone mineral density within the first year post-transplant will be assessed. Three DXA analyses will be performed, one within the first four weeks post-transplant, one five months and one twelve months after kidney transplantation.The objective is to evaluate the influence of vitamin DClinicalTrials.gov NCT00752401 Two hydroxylation steps in the metabolic activation of vitamin D3 are necessary to convert it into its physiologically active form 1,25-dihydroxyvitamin D3 . Vitamin D3 is first hydroxylated in the liver to 25-hydroxyvitamin D3  and subsequently converted to 1,25-dihydroxyvitamin D3 by 1α-hydroxylase in the kidney [Vitamin D is a steroid hormone important for the regulation of calcium homeostasis and bone metabolism. There are two ways of obtaining vitamin D, either from the diet or by cutaneous production. Exposure of the skin to ultraviolet light induces the conversion of 7-dehydrocholesterol to previtamin D3 is considered the most reliable indicator of vitamin D status as it reflects an individual's dietary intake of vitamin D on the one hand and the endogenous production in the skin on the other hand [3 is more important to achieve adequate body stores than dietary intake due to the fact that vitamin D is found in a limited variety of foods, mainly oily fish. Casual exposure to sunlight supplies about 90% of the vitamin D requirement for most people [25-hydroxyvitamin D3 decreases in elderly subjects compared to younger adults [3 ≤ 37 nmol per liter [Especially, the elderly and chronically ill are at high risk to develop vitamin D deficiency due to poor dietary vitamin D intake on the one hand and their often modest outdoor activities and the lack of exposure to sunlight on the other hand ,5. Moreoer liter .3 levels showed that only about 15% of kidney transplant recipients have adequate vitamin D levels at the time of transplantation [3 levels in 31 kidney transplant recipients who all protected themselves from sun exposure compared with an age- and gender-matched control group at the end of winter. Serum 25-hydroxyvitamin D3 levels were significantly lower in kidney transplant recipients compared with controls, with 10 out of 31 having undetectable serum 25-hydroxyvitamin D3 levels [Accordingly, a high prevalence of vitamin D deficiency was found in predialysis patients with moderate and severe chronic kidney disease as well as in patients undergoing hemodialysis ,7. Analyantation ,9. Afterantation . Consideantation  they are3 levels . For lon3 levels .Considering the high prevalence of vitamin D deficiency in kidney transplant recipients the investigation of vitamin D substitution is of particular interest and relevance to renal transplant patients.3, due to a reduction in renal parenchyma leading to decreased 1α-hydroxylase available for converting 25-hydroxyvitamin D3. Therefore, active vitamin D supplements have effectively been used in order to maintain calcium homeostasis and to decrease the risk of hyperparathyroidism in patients with chronic kidney disease.Chronic kidney disease is accompanied by decreased production of the active form of vitamin D, 1,25-dihydroxyvitamin D3 serves as a substrate for 1α-hydroxylase not only in the kidney but also in several extra renal tissues. For instance, there is evidence that immune cells express 1α-hydroxylase and are therefore able to synthesize active vitamin D locally. Thus, these tissues are dependent on adequate 25-hydroxyvitamin D3 substrate for adequate local 1,25-dihydroxyvitamin D3 production [However, it is still important to correct calcidiol deficiency as 25-hydroxyvitamin Doduction .As such, vitamin D is thought to be a potent immune modulator.3levels are associated with an increased risk of infections [Besides its classical actions in calcium homeostasis and bone metabolism vitamin D plays an important role in the immune system ,16. Due fections ,19.3 is able to shift the T-cell response towards a T-helper 2 phenotype which is considered to be protective in transplantation [3 are able to inhibit T-cell proliferation and their cytokine production (e.g. Interferon-γ), thus, inducing regulatory T-cells with suppressive activity rather than effector T-cells [Calcitriol acts as an inhibitor of the adaptive immune system with T-lymphocytes and antigen-presenting cells as key targets . On the antation  and on t T-cells ,23. Thes T-cells ,25.In clinical trials, a beneficial impact of calcitriol administration in kidney transplant recipients on graft function, overall graft survival and incidence of acute rejection episodes was demonstrated -29. HoweIn several studies beneficial effects of active vitamin D on progression of chronic kidney disease were documented, either by interfering with the transforming growth factor beta 1 (TGF-β1) signaling pathways or with the renin-angiotensin-aldosterone system, both considered to be important modulators of the progression of renal insufficiency ,32.3 was shown to function as a negative regulator of the renin-angiotensin system in animals [1,25-dihydroxyvitamin D animals . Recentl animals .3 was reported to attenuate the development of glomerulosclerosis and progression of albuminuria accompanied by a lower expression of TGF-β1 in the kidney [In diverse rat models of kidney disease 1,25-dihydroxyvitamin De kidney ,36.3. Calcitriol reduces podocyte loss and changes of podocyte foot process structure leading to a decreased albumin excretion [Furthermore, the podocyte was identified as an important target for the action of 1,25-dihydroxyvitamin Dxcretion ,38.A recent clinical trial indicated that paricalcitol reduces proteinuria in patients with chronic kidney disease. Agarwal et al. analyzed the effect of paricalcitol on proteinuria in 195 chronic kidney disease patients stage 3 and 4 compared to placebo. After six months 51% of the paricalcitol treated subjects compared to 25% of the placebo treated subjects were found to have a reduction in proteinuria independent from the use of inhibitors of the renin-angiotensin-aldosterone system .3 not only was reported to prolong graft survival but also to prevent histological changes typically found in chronic allograft nephropathy by altering TGF-β1 and matrix regulating molecules [TGF-β1 is furthermore considered to play an important role in the development of chronic allograft nephropathy. Chronic allograft nephropathy is characterized by interstitial fibrosis and glomerulosclerosis and frequently leads to late allograft loss in kidney transplantation . There iolecules . In clinolecules .3 obviously has an impact on innate and adaptive immunity, both responsible for acute rejection and chronic allograft nephropathy which is often observed to start only a few months after kidney transplantation.1,25-dihydroxyvitamin DThe proposed study is designed to evaluate whether cholecalciferol substitution in vitamin D deficient kidney transplant recipients has beneficial effects upon the post-transplant outcome compared to placebo. As a primary outcome we will evaluate the impact of cholecalciferol on- graft function  one year after kidney transplantation- the incidence of acute rejection episodes within the first year after kidney transplantation, and- the frequency and severity  of infections within the first year after kidney transplantation.2).As a secondary objective we will assess whether cholecalciferol substitution influences the common decline of bone mineral density within the first year after kidney transplantation  analyzedMoreover, courses of calcium levels will be observed in order to assess the safety of cholecalciferol administration at 6800 International Units (IU).3 < 50 nmol per liter. In case there are less than 60 subjects included in the trial after one year it is intended to recruit two other transplant centers.The VITA-D study is a single-center, randomized, placebo-controlled parallel group study with blinding of patients, study staff, and outcome assessors. Patient recruitment, kidney transplants, postoperative care, and follow-up are conducted at the Medical University of Vienna. The study is planned to start in January 2009. There are about 150 kidney transplants performed annually at the Medical University of Vienna. Approximately 30% of all kidney transplant recipients are expected to be vitamin D deficient defined as 25-hydroxyvitamin D3 < 50 nmol per liter) with 100 subjects in each study group. Inclusion and exclusion criteria are detailed in the Appendix. The recruitment phase will last approximately four years with a follow-up period of one year for each subject. A flowchart of the VITA-D study is shown in Figure 1 = 1,9 mg per deciliter with sigma, the common standard deviation, estimated to be σ = 0.5. An expert panel evaluated that a difference in serum creatinine levels of 0,2 mg per deciliter would represent a significant clinical improvement for the patient. Assuming that α = 0.05 and the power = 0.80 a sample size of 99 for each sample was calculated.The study sample will consist of 200 kidney transplant recipients found to be vitamin D deficient  or a placebo solution. Treatment starts on day five after kidney transplantation. Cholecalciferol will be administered at a dose of 6800 IU daily over a time period of one year.Kidney transplant recipients found to have 25-hydroxyvitamin D3 that may occur is the rise in blood levels of calcium. In case of hypercalcemia the dose can be reduced immediately as serum calcium levels are monitored routinely at close intervals . At serum calcium levels > 2,65 mmol per liter vitamin D3 administration will be reduced to 3600 IU per day. If calcium levels persist above 2,85 mmol per liter over a period of four weeks vitamin D3 administration will be discontinued and restarted when serum calcium levels declined to ≤ 2,65 mmol per liter with only 3600 International Units per day. If calcium levels remain high this may be attributed to a persistent or transient hypercalcemia due to hyperparathyroidism as transient hypercalcemia is observed in 20–50% in kidney transplant recipients. Thus, the subject will be tested with ultrasound and 99mTc-methoxyisobutyl isonitrile (MIBI) scan. However, these subjects will not be excluded from the study.The only adverse drug reaction caused by vitamin DPrior to kidney transplantation the administration of all active vitamin D supplements which are prescribed to almost all patients on dialysis will be discontinued.Most kidney transplant recipients are administered a triple immunosuppressive therapy consisting of either mycophenolatmofetil or mycophenolat-natrium, tacrolimus, and prednisone. Steroid boli, monoclonal, and polyclonal antibodies are administered to treat acute rejection episodes.3 analyses.Prior to transplantation patients will be recruited from the acute dialysis ward. There, and previous to kidney transplantation, blood samples will be taken of all kidney transplant candidates as a matter of routine, including a blood sample for 25-hydroxyvitamin D3 levels below 50 nmol per liter information about the VITA-D study, its purpose, putative benefits, and possible risks will be given to the patient and he/she will be invited to participate in the trial. All subjects will be informed that the participation in the trial is voluntary and that the subject may refuse to participate or withdraw from the trial at any time without giving reasons, without loss of benefits. Kidney transplant recipients who do not agree to participate in the study will receive standard care.If a kidney transplant recipient is found to have 25-hydroxyvitamin DDemographic and baseline data will be collected of all participants within the first five days after transplantation .3 therapy or placebo.Kidney transplant recipients who agreed to participate in the study will be randomized to receive either oral vitamin DKidney transplant recipients are treated in hospital between two and four weeks on average. Within this time period blood samples are taken daily as a matter of routine, including analyses of serum creatinine, C-reactive protein levels and calcium levels.The first DXA  analysis will be performed within the first four weeks after transplantation depending on postoperative complications. However, we aim at performing it within the first two weeks.After discharge kidney transplant recipients are seen once a week within the first four months after transplantation and are thereafter scheduled for visits every two to four weeks within the first year after transplantation. Blood samples are taken at every visit at the nephrological outpatient department.3 levels will be analyzed in order to check the response to treatment. In addition, eating habits and lifestyle  will be evaluated by means of a questionnaire.25-hydroxyvitamin DThe second DXA analysis will be performed.The third DXA analysis will be performed.A summary of all study procedures is shown in Figure Baseline data will be expressed as mean values ± standard deviation for symmetrically distributed continuous variables, as median and 25th and 75th percentiles for asymmetrically distributed continuous variables, and as absolute and relative frequency for categorical variables.3 at baseline, and variables which, despite randomization, exhibit large group differences at baseline. ANCOVA will also be applied to the area under the curve of repeated measurements on serum creatinine, CRP, and bone mineral density during the first year of follow-up, using the same set of covariates. Categorical variables (number of acute rejection episodes and infections within the first year after transplantation) will be analyzed using χ2-test or fisher's exact test, as appropriate.Serum creatinine values after one year (the primary endpoint) will be compared between the groups by analysis of covariance (ANCOVA), taking into account as covariates creatinine value at randomization, 25-hydroxyvitamin DAll analyses will be performed using an "efficacy subset analysis". Patients are included in this analysis if the study medication has been administered for more than seven days.Moreover, courses of calcium levels will be described. Furthermore, the Bonferroni-Holm correction will be performed to preserve the global significance level of 5%.Statistical analysis will be performed with SPSS.An interims analysis will be performed after 6 months of follow-up for every subject. Within this analysis glomerular filtration rates between the two groups six months after transplantation and the incidence of acute rejection episodes within six months after transplantation will be analyzed. In case the interims analysis shows a significant difference between the two groups the study will be discontinued prematurely. The strict significance level of 0.001 for interim analysis requires no modification of the significance level for the main analysis . The intThe proposed study will be conducted in accordance with the Declaration of Helsinki, the Austrian law and subscribes to the principles outlined in the International Conference on Harmonization on Good Clinical Practice, 2002. Approvals were obtained by the Ethics Committee of the Medical University of Vienna and the Vienna General Hospital (Reference Number 213/2008) and the Austrian regulatory authority . Furthermore, the study has been registered in a public clinical trial database .3 at a dose of 6800 IU per day is not expected to pose any risks of severe adverse health effects. Vitamin D intoxication is extremely rare and can only be caused by intake of excessively high doses. Doses of more than 50 000 IU per day can raise 25-hydroxyvitamin D3 levels to more than 370 nmol per liter and are associated with hypercalcemia and hyperphosphataemia [3 is likely to pose no risk of adverse effects, the recommended upper intake level is set at 10 000 IU per day [3 is less for patients with renal disease than for the rest of the population [Considering current clinical trial data -48 the ahataemia . Howeverhataemia  is set apulation .3 in kidney transplant recipients showing vitamin D deficiency. We will investigate whether or not vitamin D3 has beneficial effects upon graft function, the incidence of acute rejection episodes, and frequency and severity of post-transplant infections. If vitamin D3 substitution proves to have no relevant disadvantages and if effectiveness can be demonstrated then clinical practice and routine supplementation of vitamin D3 in vitamin D deficient kidney transplant recipients may be intensified.To date, clinical trials investigating vitamin D and its immunomodulatory properties within the transplant setting have been sparse, only a few small trials are available. We report the rationale, design, and methodology of the VITA-D study, the first prospective, randomized, placebo-controlled trial focusing on the immunomodulatory and renoprotective properties of vitamin DThe study medication is provided by Fresenius Kabi Austria Gmbh, the marketing holder of cholecalciferol in Austria. However, the authors do not receive any reimbursement or financial benefits from the company and declare that they have no competing interests.UT made substantial contributions to the conception and design of the study and was essentially involved in drafting the study protocol and the manuscript.GH participated in the design of the study by giving advice on statistics and will be involved in the statistical analyses.RS will be in charge of the production, labeling and blinding of the investigational products.TP participated in the design of the study by giving advice on laboratory procedures and will be involved in the laboratory analyses.FK participated in the design of the study and helped to draft the manuscript.FM participated in the design of the study and its coordination and helped to draft the manuscript.WH participated in the design of the study and its coordination and helped to draft the manuscript.KB is the principal investigator, responsible for recruitment and trial coordination. She developed the study idea and made substantial contributions to conception and design. Moreover, she was involved in drafting and revising the study protocol as well as this manuscript.All authors will participate in the implementation or analysis of this study and approved the final manuscript.Age > 18Deceased donor kidney transplant recipientsOnly kidney transplant recipients3 < 50 nmol per literVitamin D deficiency defined as 25-hydroxyvitamin DRe-transplantation for the second time if the patient is highly immunized and therefore included in the aphaeresis programRe-transplantation for the third or further timeSignificant impaired intestinal resorptionHistory of inflammatory bowel disease: Crohn's disease, Ulcerative ColitisPrevious gastrectomy, small bowel or large bowel resection, intestinal bypass surgerySevere liver disease: cirrhosisHIV positive


To quantify the likely impact of recent WHO policy recommendations regarding smear microscopy and the introduction of appropriate low-cost fluorescence microscopy on a) case detection and b) laboratory workload.An audit of the laboratory register in an urban hospital, Lilongwe, Malawi, and the application of a simple modelling framework. The adoption of the new definition of a smear-positive case could directly increase case detection by up to 28%. Examining Ziehl-Neelsen (ZN) sputum smears for up to 10 minutes before declaring them negative has previously been shown to increase case detection (over and above that gained by the adoption of the new case definition) by 70% compared with examination times in routine practice. Three times the number of staff would be required to adequately examine the current workload of smears using ZN microscopy. Through implementing new policy recommendations and LED-based fluorescence microscopy the current laboratory staff complement could investigate the same number of patients, examining auramine-stained smears to an extent that is equivalent to a 10 minutes ZN smear examination.Combined implementation of the new WHO recommendations on smear microscopy and LED-based fluorescence microscopy could result in substantial increases in smear positive case-detection using existing human resources and minimal additional equipment. There are an estimated 9.3 million new cases of tuberculosis (TB) each year, with the majority occurring in low- and middle-income countries (LMICs) The World Health Organization (WHO) has recently made policy recommendations relevant to sputum microscopy in settings with well-functioning External Quality Assessment (EQA) schemes. The recommendations include lowering the thresholds for: a) classifying a smear as positive from ≥10 Acid Fast Bacilli (AFB)/100 High Power Fields (i.e. 1+ grade) to 1 AFB per smear , and b) classifying a patient as a smear-positive case (from two positive smears to one) The WHO, recognizing the minimal additional yield provided by the third specimen, has also recommended that, in settings with high laboratory workloads and limited human resources, the minimum number of sputum specimens to be examined in the investigation of suspected PTB could be reduced from three to two The sensitivity of smear microscopy is reported to be variable even in epidemiologically similar settings. The amount of time spent examining smears is critical but seldom reported. Adequate time must be invested if paucibacillary cases are to be detected. International guidelines recommend a 5 to 10 minute examination, equivalent to the examination of between 100–300 high-power fields (HPF), before declaring a smear to be negative It is recognized that fluorescence microscopy (FM) could also increase case detection and reduce laboratory workloads. A recent systematic review indicates that FM may increase sensitivity of DSSM by around 10%. Perhaps more importantly, the examination of fluorochrome-stained smears can be achieved in 25% of the time taken to examine Ziehl-Neelsen (ZN) stained smears (with comparable specificity) Both the recent policy changes and LED-FM seem suitable for diagnostic services in Malawi, which have endured long-standing and severe human resource shortages Retrospective laboratory data from an urban hospital in Malawi was used to model:the increase in smear-positive case detection that might be expected to stem directly from adoption of the WHO-recommended lowered threshold for definition of a positive smear and a smear-positive case;the reduction in laboratory workload that might be expected from adopting a two-specimen strategy for investigating suspected pulmonary TB andthe potential for examining ZN stained sputum smears for the recommended 10 minutes  with the existing designated laboratory staff.This is the first attempt to model the potential impact of these interventions that has been reported as far as we are aware.This retrospective analysis used data from the laboratory register of Bwaila Hospital in Lilongwe District, Central Malawi Sputum smear microscopy is used to investigate patients with ≥3 weeks cough (TB suspects) and to monitor response to TB treatment (follow-up patients). TB suspects are asked to submit three sputum specimens collected as spot-morning-spot over two days. Follow-up patients are usually monitored during the second and fifth months of treatment by examining two sputum specimens on each occasion. Specimens are submitted to the laboratory from Monday to Friday and records are kept in standardized laboratory registers. Data extracted from the register included the date the sputum was received, sex, patient category (i.e. TB suspect or follow-up), the number of specimens examined and result of smear microscopy. In order to minimize disruption, data were extracted from the most recent laboratory register that was not in daily use. Data were obtained for 24 April - 23 October 2006 (6 months). The data were used to calculate: the numbers, sex and categories of patients investigated; the number of specimens submitted; the number of follow-up patients with positive smears; the number and sex of TB suspects with positive smears; and the number and sex of smear-positive cases as then defined. An international system of laboratory workload measurement (Welcan) was used to estimate the workload and staff requirements of actual practice during the period The Welcan system assigns a value to laboratory tests in terms of hands-on staff time required. Hands-on time may be defined as time that a staff member is actively working. Hands-on time does not, for example, include time while smears are being passively stained (eg auramine staining for FM) but does if the stain needs to be heated periodically (eg carbol fuchsin staining for ZN). The Welcan system is based on observation and timing of procedures in routine laboratories and takes account of batching of smears etc. The system assigns a value of 15 minutes to the complete processing of a single sputum specimen using ZN smear microscopy (from specimen receipt to reporting). In the model, 5 of these minutes were assigned to the non-microscopy component of the processing and a maximum of 10 of these minutes would be assigned to the microscopic examination of the ZN smear until the result was declared. All 10 minutes would be required to declare a smear negative, and proportionately less for a 3+, 2+, 1+ and scanty positive smear. The overall workload would thus be affected by the proportion of positive smears and their grades, as these take less time to examine. To estimate the workload more realistically in the model, shortened examination times were be attributed to positive ZN smears . As data on the grades of follow-up patients smears were not collected from the laboratory register, an average of 2.5 minutes microscopy time was attributed for each positive smear of follow-up patients. It is assumed that the 5 minutes non-microscopy component of the Welcan value for ZN smears is fixed as clerking and processing of the samples is not affected by specimen positivity and there is little opportunity for expediting the process.The Welcan system assigns a value of five minutes to the complete processing of a single sputum specimen using fluorescence microscopy (from specimen receipt to reporting). In the model, 2.5 of these minutes were assigned to the non-microscopy component of the processing and a maximum of 2.5 of these minutes would be assigned to the microscopic examination of the FM smear until the result was declared (i.e. 25% of the recommended 10 minute ZN microscopy time). All 2.5 minutes would be required to declare a smear negative, and proportionately less for a 3+, 2+, 1+ and scanty positive smear. The overall workload would thus be affected by the proportion of positive smears and their grades, as these take less time to examine. To estimate the workload more realistically in the model, shortened examination times were be attributed to positive FM smears . As data on the grades of follow-up patients smears were not collected from the laboratory register, an average of 1.5 minutes microscopy time was attributed for each positive smear of follow-up patients.The number of staff required for the workload arising from the different interventions was then calculated, using reported details of district laboratory working patterns in Malawi Permission for conducting the audit was obtained from the Lilongwe District Health Office and the National Tuberculosis Control Programme (NTP). General measures are provided in all NTP facilities to ensure patient confidentiality. Data collected during this audit did not include any personal identifiers. The Malawi National Health Science Research Committee provides general oversight and approval for collection and use of programmatic data for monitoring and evaluation and improvement of service delivery. The work described in this report was conducted under this arrangement.A total of 2449 patients were registered with a median of 86 patients (range 48–178) and 239 (range 140–482) smears examined per week. Of these, 1920 patients were TB suspects and 529 follow ups. In total 6,796 smears, 5,730 (84.3%) from TB suspects and 1,066 (15.7%) from follow-up patients, were examined. Results from thirty smears from TB suspects were not recorded . Eight follow-up patients had 3 smear examinations recorded possibly in error. Five hundred and sixty one (9.7%) of the 5,730 smears from TB suspects and 49 (4.6%) of the 1,066 smears from follow-up patients had ≥1 AFB resulting in 201 (10.5%) and 26 (4.9%) of the TB suspects and follow up patients being smear-positive. A median of 7 (range 3–13) TB suspects and 1 (range 0–3) follow up patient were positive per week and 19 (range 9–38) and 2 (range 0–6) smears from TB suspect and follow up patients were positive each week.In total, 1099 (57.2%) of the 1920 TB suspects and 322 (60.9%) of the 529 follow up patients were male. Among the TB suspects, 128 (11.6%) of 1099 males and 73 (8.9%) of 819 females had ≥ one smear with ≥1 AFB (p = 0.06), while among follow-ups, 15 (4.7%) of 322 male and 11 (5.3%) of 206 female were positive (p = 0.8). Discrepancies between numbers are accounted for by patients for whom no sex was recorded or whose smears were not graded.The numbers of TB suspects identified as having smear-positive PTB stratified by the case definitions and sex are shown in If the 1,920 TB suspects had submitted three sputum specimens 5,760 smears should had been examined. Investigating TB suspects by examining only 2 specimens would reduce the actual workload from 5730 to 3840 smears. The first smear to be positive was the first spot smear in 125 (98%) of the 128 males and 70 (97%) of the 72 females, the remaining 3 (2%) male and 2 (3%) female patients were identified as smear positive on the morning smear. If only one smear with ≥1 AFB were considered sufficient for diagnosis, 195 of 200 patients, being positive on the first smear, would not need examination of the second smear. This would reduce the workload by a further 195 smears to 3645. In accordance with current recommendations, the 529 follow-up patients would continue to be monitored with two smears regardless of the results of the first smear requiring 1,058 smears. The total number of smears therefore would have been reduced from 6788 to 4703 (a reduction of 31%).Trained laboratory workers at district hospital level are estimated to work 1240 hours per year (23.8 hours/week) The workloads were re-calculated for ZN and FM if a two specimen strategy were adopted for the investigation of TB suspects, as shown in Finally, the workloads were re-calculated for ZN and FM assuming the adoption of a two specimen strategy for the investigation of TB suspects and that one positive smear is sufficient to define a smear-positive case, in accordance with the newly recommended definition. In accordance with current recommendations, examination of two smears was still required for follow-up patients regardless of the result of the first smear. The adoption of this approach would result only in a slightly reduced time needed for microscopy since the second specimen from TB suspects would still have been registered and a smear prepared, fixed and stained. Overall, this approach would only reduce the workload marginally for ZN, from 1.89 to 1.87 FTHOE, and not at all for FM (FTHOE remains 0.64).At the time of this audit, two positive smears were required to define a smear-positive case based on microscopy alone in Malawi and the required grading of the smears was not stated in its national guidelines. A person with a single positive smear could be categorized as a smear-positive case if they had radiographic abnormalities consistent with TB, or had a positive TB culture. TB culture was not routinely available for the diagnosis of new cases and many diagnostic centres did not have ready access to chest radiography. The situation is the same in 2009. This audit indicates that adoption of the revised WHO smear-positive case definition could significantly increase case detection in men and women by up to 28% at centres without access to chest radiography or TB culture depending on the grade of smear currently accepted as positive. In contrast to a prospective study conducted in Kenya, this retrospective analysis found that the increase in case detection may be significantly higher in men than in women This audit also demonstrates that the laboratory evaluated is overwhelmed with requests for sputum smear microscopy relative to its human resources and equipment. Similar smear microscopy workloads and human resource shortages were described from Ntcheu District Hospital in Malawi over 10 years ago. The laboratory workload and available human resources at Bwaila Hospital are likely to be similar to those at other district hospital laboratories in Malawi and neighbouring countries Reducing the number of specimens examined in the investigation of TB suspects from three to two reduced the workload by 31%., but it still would require a total of 1.89 FTHOE staff if ZN microscopy was used. This most likely represents 4 staff employed, with 3 staff (each with a microscope) employed for the microscopy component alone (1.23 FTHOE). In all approaches using ZN smear microscopy the time taken for the microscopy component, and the professional laboratory staff (Skill Level II) time required, was around twice the time taken for the non-microscopy component and the general staff (Skill Level I) time. This has implications for service costs.The introduction of fluorescence microscopy to an approach in which three specimens from TB suspects are examined considerable reduced workload and particularly the time required of Skill Level II (microscopy) staff. A total FTHOE staff equivalent of 0.89 of which 0.44 FTHOE would be Skill Level II probably brings the total employed staff required to around 2–3 staff. The combination of reducing the number of specimens examined in the investigation of TB suspects and the adoption of fluorescence microscopy brings the clearest indication that workload could be reduced to the levels at which the existing staff employed on smear microscopy at Bwaila Hospital could provide a quality smear microscopy service. A total of 0.64 FTHOE staff split equally between Skill Levels I and II (0.32 FTHOE) brings the total employed staff required to two (one at Skill Level 1 and one at Skill Level II).The combination of adopting the new WHO recommendations on smear microscopy and introducing fluorescence microscopy can be expected to considerably reduce workload and almost double smear-positive case detection. Furthermore this increase in case detection would be achieved with the existing human resources available and minimal additional equipment.The adoption of the new WHO smear-positive case definition directly increased case detection (at least by ZN), but the obviation of confirmatory smears would have a negligible impact on workload regardless of whether ZN or FM were used.Interestingly, the laboratory was equipped with a functional conventional fluorescence microscope, which was infrequently used. Only one member of staff was trained to use it. There are many reports of conventional fluorescence microscopes being poorly accepted by laboratory staff in LMICs The new LED-based FM systems come as complete microscopes or as adaptors for existing light microscopes A number of challenges need to be overcome before LED-FM can be implemented widely in NTP diagnostic centres, including the large-scale training that would be required. The most challenging obstacle is the need to develop and validate adequate EQA systems for fluoresce microscopy


To compare medical students on a modern MBChB programme who did an optional intercalated degree with their peers who did not intercalate; in particular, to monitor performance in subsequent undergraduate degree exams.This was a retrospective, observational study of anonymised databases of medical student assessment outcomes. Data were accessed for graduates, University of Aberdeen Medical School, Scotland, UK, from the years 2003 to 2007 (n = 861). The main outcome measure was marks for summative degree assessments taken after intercalating.rd year) performance in matching exam type, having done an IC degree was significantly associated with attaining high (18–20) common assessment scale (CAS) marks in three of the six degree assessments occurring after the IC students rejoined the course: the 4th year written exam (p < 0.001), 4th year OSCE (p = 0.001) and the 5th year Elective project (p = 0.010).Of 861 medical students, 154 (17.9%) students did an intercalated degree. After adjustment for cohort, maturity, gender and baseline (3Intercalating was associated with improved performance in Years 4 and 5 of the MBChB. This improved performance will further contribute to higher academic ranking for Foundation Year posts. Long-term follow-up is required to identify if doing an optional intercalated degree as part of a modern medical degree is associated with following a career in academic medicine. Traditionally, about one-third of UK medical students undertake a year additional to the basic five year undergraduate course to intercalate a degree. However, this figure varies widely across medical schools , from 5% to 100% [There appear to be a number of benefits to doing an intercalated degree. A longitudinal study of all UK medical school graduates in 1996 and 1997 found students who had taken an intercalated degree had higher strategic and deep learning scores and lower surface learning scores than those who had not . IntercaHowever, other factors seem to discourage medical students from intercalating. One potential factor is the norm of the medical school: if these degrees are perceived to be only for high achievers, then many students will not consider this option . Where s 2nd August 2007). Ranking also depends on the average of the student's performance over the medical degree: better performance leads to higher ranking which is beneficial when applying for competitive posts. Thus any short-term benefit to intercalating, in terms of improved performance in subsequent undergraduate degree exams , may attract students to doing an intercalated degreeUK Medical training has changed in recent years. The introduction of Modernising Medical Careers  encouragUnfortunately, those studies which have examined intercalating student performance on rejoining the medical course provide conflicting evidence ,12, invoThis study aimed to compare medical students on a modern MBChB programme who did an optional intercalated degree with their peers who did not intercalate; in particular, to monitor performance in subsequent undergraduate degree exams.The context of this study was a long-established medical school with a distinguished research history. The school is relatively small . At the time of data collection, the medical degree was a 5-year programme with a mostly undergraduate intake. The programme emphasised early clinical experience; had a vertical communication theme; was problem-oriented, with a lecture- and Student Selected Component-based approach to teaching and learning. Comparison of career choices by medical school in graduates of 1999 and 2000 indicated that Graduates from the school under study were more likely to choose General Practice, Anaesthetics, A&E and other medical specialties, and less likely to choose surgical specialties, as their first choice of long-term career than the national average .rd and 4th year of the five year basic programme. Students applied mid-3rd year, then must have attained a minimum grade in 3rd year  to be accepted to intercalate. All intercalating (IC) students complete a core course on foundations of medical research, a second set of core topics that are related to clinical or laboratory-based research (depending on the student's research project), then a 20-week research project which is written up as a thesis.The study subjects were all University of Aberdeen MBChB students who graduated in the years 2003 to 2007. At the time of data collection, the Aberdeen MBChB intercalated degree programme was placed between 3Data on gender, age, funding status (home or overseas), any previous degree and marks are routinely collected during the selection and degree assessment processes.Marks are collected in the form of the common assessment scale (CAS), a 21-point non-linear scale from 0–20, used for all assessments at the University of Aberdeen.Only marks for summative degree assessments were examined in this study (see Table rd year) performance in matching exam type; cohort; maturity and gender. Note that previous degree was not included as a covariate due to problems of collinearity with the variable 'maturity' since the vast majority of the mature students had a previous degree. The Nagelkerke pseudo R2, was also documented. This can take a value of between 0 and 1 and is a marker of the improvement in goodness of fit of the current model over a model containing just the intercept term. If the independent variables in a model perfectly predicted the outcome, then the Nagelkerke pseudo R2 would equal 1. Due to the relatively high number of statistical tests performed throughout the manuscript, and the resulting increase in likelihood of a type 1 error , a more stringent value of p ≤ 0.01 was used to denote statistical significance throughout all analyses.SPSS for Windows, Version 16.0 was used for data storage and analysis. The Mann-Whitney test was used to compare the distribution of age across intercalated groups since it followed a non-Normal distribution. Categorical data were described as percentages and associations between intercalation and CAS band were compared using the chi-square test. The Aberdeen CAS system is such that mean scores were not available and therefore effect sizes could not be estimated. Multinomial logistic regression was used to explore the association of intercalating with student performance in the fourth and fifth year assessments. The regression procedure shows the odds of attaining a particular CAS mark band in a specific exam for intercalating versus non-intercalating students. Next, to account for the potential confounding of those who chose to take an intercalated degree after third year being better and/or more mature students from the outset, the following covariates were simultaneously included in each model: baseline ; there were 59 (7%) overseas-funded students and 92 (11%) graduates. One hundred and fifty-four (17.9%) students did an intercalated degree, a median of 31 students per year (range 29–34).Table nd year did not sit these exams.Tables th and 5th year, the 4th year written final .Table rd year OSCE (OSCE3), which takes place just before the IC year, IC students performed significantly better than their non-IC colleagues (p = 0.003). No significant associations were found for the 4th or 5th year OSCEs (OSEC4 p = 0.015 and OSCE5 p = 0.025) although the general trends were in the same direction.In the 3rd year Community Course III  and 5th year Elective project  was also seen.A significant association between student performance and intercalating in the 3rd year) performance of the matching exam type, doing an IC degree was significantly associated with attaining CAS marks of 18–20 in several of the 4th and 5th year exams. Students with an IC degree were over five times more likely than non-IC students to attain CAS marks of 18–20 in the 4th year written Specialist Clinical Practice exam . Similarly, IC students were almost three times more likely than non-IC students to attain CAS marks of 18–20 in the 4th year OSCE (OSCE4) and they were more than five times more likely than non-IC students to attain CAS marks of 18–20 in the 5th year Elective Project (JP5:1).Table This study aimed to identify if students on a modern degree programme who did an optional intercalated degree performed better in subsequent undergraduate degree exams than their peers who did not intercalate. When we adjusted for performance in the early years of the course along with maturity and other covariates, intercalating was associated with gaining high CAS marks in three of the six degree assessments which took place after rejoining the course. These assessments reflected the range of assessment methods: written, clinical and essay-based.We know that intercalating confers benefits in terms of gaining additional points in the academic ranking system for applications for FY1 posts. Our data indicates that intercalating was also associated with additional benefits in terms of improved performance in Years 4 and 5 of the MBChB. Improved performance will further contribute to higher academic ranking.The relatively low numbers of students who choose to intercalate in Aberdeen (just under 18%) suggests that our students are not aware of these benefits – intercalating may become more attractive if they become so. Moreover, the proportion of Aberdeen students intercalating is well under the average proportion intercalating across all UK medical schools of 36% . Thus, wIntercalating students do significantly better in their final year elective project. This is a research project so it is not surprisingly that IC students, who had received more training and practice in the skills required to produce this kind of work than those who did not intercalate , do bettWhy do students who intercalate do better once they rejoin the medical course? We suggest this is probably due to a combination of maturity  and gainrd year of the matching exam type and therefore our overall findings are not simply a reflection of a higher academic ability of the IC students.Given that entry into the Aberdeen intercalated degree programme is based on attainment in third year, we expected IC students to do better than their colleagues on early exams. This was indeed apparent on several of the written exams. We propose that this might suggest that those who chose to do an intercalated degree are more academically-inclined than the average student. Thus, rather than an intercalated degree encouraging entry into academic medicine -6, it mard year clinical (OSCE) exam. This pattern was maintained when the IC students rejoin the MBChB programme. IC students have no clinical contact in their intercalating year, so any improvement is likely due to maturity [Intercalating students did better than their colleagues on the 3maturity  and/or bmaturity .Younger students were more likely to do an IC degree. This may be associated with the fact that many older students were graduates, or simply with the fact that more mature students may have financial and/or family commitments, beyond those of the average school-leaver, which make an additional year of study difficult. While one would not expect a graduate to do an IC degree, we were initially surprised that no overseas student had ever intercalated. The most probable explanation for this is additional cost, especially in light of the increased amount overseas students pay in fees.This is the first study exploring the short-term benefits, in terms of improved performance on subsequent undergraduate assessment, of intercalating in a population of students doing a modern (post Tomorrow's Doctors ) MBChB iThe design of this study does not allow for exploration of whether intercalating predisposes a medical student towards an academic career, or whether those who are predisposed towards an academic career are more likely to intercalate. If the former, the short-term gains from intercalating identified in this study may attract more medical students to intercalate, and hence, more graduates to academic medicine.In conclusion, intercalating has been reported to be beneficial to learning to learn and to an academic medicine career. Having an Honours degree contributes to academic ranking for Foundation Year applications in the UK. This study adds that intercalating was associated with improved performance in degree assessments after students rejoined the MBChB. Improved performance was measurable on a range of assessment methods. It may be that the short-term gains from intercalating identified in this study may attract more medical students to intercalate. We suggest this study be replicated in other UK medical schools to explore whether the short-term benefits associated with an intercalated degree that we identified at Aberdeen, are widespread. Long-term follow-up is required to identify if doing an optional intercalated degree as part of a modern medical degree is predictive of following a career in academic medicine.The authors declare that they have no competing interests.HS and JC had the original idea for this study. All authors were involved in the design and execution of the project. AM and AJL carried out the statistical analysis. JC wrote the first draft of the paper. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Alzheimer's disease (AD) is a progressive neurodegenerative disorder involving variations in the transcriptome of many genes. AD does not affect all brain regions simultaneously. Identifying the differences among the affected regions may shed more light onto the disease progression. We developed a novel method involving the differential topology of gene coexpression networks to understand the association among affected regions and disease severity.We analysed microarray data of four regions - entorhinal cortex (EC), hippocampus (HIP), posterior cingulate cortex (PCC) and middle temporal gyrus (MTG) from AD affected and normal subjects. A coexpression network was built for each region and the topological overlap between them was examined. Genes with zero topological overlap between two region-specific networks were used to characterise the differences between the two regions.Results indicate that MTG shows early AD pathology compared to the other regions. We postulate that if the MTG gets affected later in the disease, post-mortem analyses of individuals with end-stage AD will show signs of early AD in the MTG, while the EC, HIP and PCC will have severe pathology. Such knowledge is useful for data collection in clinical studies where sample selection is a limiting factor as well as highlighting the underlying biology of disease progression. The neuropathological hallmarks of Alzheimer's disease (AD) are the accumulation of extracellular amyloid plaques and intraneuronal neurofibrillary tangles (NFT) in the brain. Certain brain regions have shown increased susceptibilities to the pathological and metabolic characteristics of AD -5. HowevOrganising genes into co-expression networks helps in comparing biological phenomena across brain regions, and obtaining a global overview of the disease, which can enable us to further understand the disease. As is well known in Quantum mechanics, while the behaviour of particles is not well understood or intuitive at the quantum level, they do behave more intuitively at the macro level. Hence, the application of systems biology methods to understand complex diseases is crucial. Gene coexpression networks can provide a view of the relationship among genes, based on their gene expression profile, in a particular condition or time or disease. Genes in a coexpression network are connected to one another based on the similarity of their expression profiles. The rationale behind this is that coexpressed genes may participate in the same pathway or form complexes ,7 that pNumerous studies have analysed the similarities in network structures and coexpression clusters -10. ReceIn this study, our objective was to identify genes with differential topology in gene coexpression networks corresponding to different brain regions and observe the difference in AD severity across regions. We first identified the differentially expressed (DE) genes between AD affected and normal controls in the entorhinal cortex (EC), hippocampus (HIP), posterior cingulate cortex (PCC) and middle temporal gyrus (MTG). Then coexpression networks for the regions were built using the common DE genes between regions. Next, we investigated the topological differences between coexpression networks, and identified the significant biological pathways of the sets of genes with no topological overlap across the region-specific networks. The significant pathways along with network gene connectivity were used to determine the association between disease stage and brain region, and the relationship between gene activity and disease severity. Figure For our analyses we used recent microarray (Affymetrix Human Genome U133 Plus 2.0) expression data obtained via laser captured microdissection and contain data from different brain regions that are either histopathologically or metabolically relevant to Alzheimer's disease (AD)  with a Ct-statistics method to identify DE genes. Within each brain region, AD affected subjects and unaffected age-matched controls were compared to identify DE genes. After obtaining four sets of DE genes, one per brain region, we identified the common (intersection) DE genes between two regions of interest. The common genes were used to create two gene coexpression networks for the pair of regions from which the common genes were selected. These sets of common genes between regions will be referred to as the 'intersection genes' in the manuscript for clarity.Probe sets were processed for differential expression (DE) by using the two-class significance analysis of microarrays (SAM) , based oSignificant biological pathways were identified using the well annotated GeneGo MetaCore™ database . MetaCort  and one gene is within the top-50 most correlated gene of the other. According to the first condition, if 3 genes are correlated to gene A with correlation coefficients equal to 0.3, 0.32, and 0.4 then they get linked to gene A. On the other hand, if the 3 genes are correlated to gene A with correlation coefficients equal to 0.3, 0.28 and 0.29, then only 1 gene would get linked to gene A. However, this scenario rarely, if at all, occurs in gene expression data with a correlation coefficient threshold of 0.3  method developed by Ruan and Zhang ,19 to coThis approach was taken to create a sparse coexpression network. Minimally complex, sparse gene networks have been shown to be more robust to perturbations and may be a constraint in shaping the evolution of gene network complexity . The CoEA coexpression network was constructed for two brain regions under study separately using the differentially expressed genes that were common to both regions, i.e. the intersection genes (see Methods). Additionally, gene coexpression networks were also created using the DE genes obtained by comparing AD affected and normal controls in order to compare affected and controls within each region. The coexpression network obtained from CoExp is a binary adjacency matrix with 0 referring to no link between two genes and 1 corresponding to a link between the genes.network1 and network2 corresponding to brain region 1 and brain region 2, respectively. Since network1 and network2 were built using the intersection genes between regions 1 and 2, the nodes in both the networks are the same, although the connections among them are different. Let each node/gene in the network be denoted as i genewhere i = 1, 2,...,m and m is the total number of nodes in the network.Let the two coexpression networks be referred to as network1 and network2, refers to the overlap of the genes connected to i genein network1 and network2, i.e. overlap of the neighbourhoods of igene. Let the genes connected to a i genein network1 be referred to as X and those in network2 be referred to as Y. The connectivity or degree of i genein network1 is d1i and that in network2 is d2i. The topological overlap (TO) for i genebetween network1 and network2 is given byTopological overlap between gene coexpression networks, i genewas considered instead of the smaller degree in order to reduce false negatives. Consider the following example. Let the neighbourhoods of i genebe X = 200 in network1 and Y = 10 in network2 with X ∩ Y = 10, then with max = 200, TO = 0.05. On the other hand, with min = 10, TO = 1. Since our aim was to identify genes with a topological difference (i.e. low TO) between coexpression networks, this gene would have been discarded  if the smaller degree was used as it would result in a high TO value. The actual amount of similarity between these two neighbourhoods is only 5% (i.e. 10/200 unique genes). The TO values in this analyses were rounded upto 4 decimal places.The larger degree of t-statistics were made to assess the significance of the zero TO genes. The actual gene co-expression networks had a lower number of zero TO genes compared to the random networks. The significance values  were calculated (with 999 degrees of freedom) using the following formula,If the topology of coexpression networks are representative of the 'biological activity' of a gene under a certain condition or in a specific region, then genes with high topological overlap between two region-specific networks, may not differ greatly in their activity in the two brain regions. However, genes with low topological overlap probably have roles/activities that are region-specific or condition-specific. As a specific case for this analysis, genes with the maximum topological difference, i.e. TO = 0  between the two networks were selected for further analyses in this study. Genes with other values of topological overlap, if properly justified, can also be considered. Comparisons against 1000 random networks  using μ1 is the mean number of zero TO genes in the real network; μ2 is the mean number of zero TO genes in randomised networks; SD is the standard deviation of the number of zero TO genes in the 1000 random networks; n1 is the number of random nets (i.e. 1000) and n2 is the number of real networks (i.e. 1).where Some studies have used the topological overlap measure for identifying metabolites that are in the same functional class , or for Significance analysis of microarrays (SAM)  was usedSix sets of intersection genes (see Methods) were obtained from six comparisons - (1) EC and HIP; 2) EC and PCC; (3) EC and MTG; (4) HIP and PCC; (5) HIP and MTG; (6) PCC and MTG . The entire list of these TO genes are provided as Additional file The aim of this study was to identify the genes with differential topology between the region-specific coexpression networks. The biological processes represented by these genes would provide an idea of the difference, if any, in disease severity among the different regions. Therefore, after the coexpression networks were built for each region, the topological overlap (TO) between the region-specific networks was calculated for each pair of regions analysed. The numbers of genes with zero TO between the coexpression networks are shown in Table SAD1 ), calcitonin gene-related peptide-receptor component protein (RCP9 ), calcitonin 1 (CALCA), cyclin-dependent kinase 2 (CDK2 ), coronin actin binding protein 1B (CORO1B), interleukin 10 (IL10 ), ninjurin 1 (NINJ1 ), oxidative-stress responsive 1 (OXSR1 ), phospholipase c eta2 (PLCH2 ), spectrin beta non-erythrocytic 1 (SPTBN1 ), plexin B2 (PLXNB2 ), synapsin III (SYN3 ), syntaxin 16 (STX16 ), lim domain and actin binding 1 (LIMA1 ), toll-like receptor 4 (TLR4 ), yy2 transcription factor (YY2 ), microtubule associated serine/threonine kinase 1 (MAST1 ), were some of the AD associated genes in the list of 300 zero TO genes between the ECnet and HIPnet, that were associated with processes such as protein transport, cytoskeletal organisation, neurotransmitter release etc. [YY2 is highly similar to the evolutionarily well-conserved zinc finger gene YY1 [BACE1 ) expression [BACE1, which was present in the list of 271 genes between HIPnet and MTGnet, is necessary for the generation of beta-amyloid peptides, the principal constituents of senile plaques, in AD subjects [TLR4 ) signalling pathway has been implicated in the clearance of beta amyloid deposits in the brain of Alzheimer's disease subjects [Although a few are mentioned here, there were many AD relevant genes in all the sets of zero TO genes , hippocampus (HIP), middle temporal gyrus (MTG), superior frontal gyrus (SFG), posterior visual cortex (PVC) and posterior cingulate cortex (PCC) . Ac-cordIt is conceivable that if a region is minimally affected by AD or AD-like (such as ageing) neurodegeneration, then inflammation or immune response related pathways will not be very statistically significant - as we have already reasoned that they are secondary processes in AD. When we analysed the DE genes of the SFG and PVC, we found this to be the case. While there were immune response pathways, there were many development and cytoskeletal related pathways. From these results we conclude that by using our method we can identify that the MTG does not seem to be as severely affected as the other 3 regions.As a comparison, we identified the significant pathways of all the intersection genes (Number of genes shown in Table CASP6 ) was present in the list of 180 zero TO genes between the MTG-PCC, 271 genes between the HIP-MTG and 192 genes between the EC-MTG. CASP6 has been associated with early AD pathogenesis [CASP6 was connected to 3 genes in the MTGnet and 11 in the HIPnet; 3 genes in the MTGnet and 24 in the PCCnet; and 21 genes in the ECnet and 6 genes in the MTGnet. Based on the large difference in connectivity between the region-specific networks, and the high connectivity in certain regions  which we have previously suggested are severely affected, we hypothesised that CASP6 acts more aggressively in the later stages of the disease. CASP6 has been implicated in the early pathogenesis of Alzheimer's disease and continues to be an active participant in the later stages of the disease [BACE2 ) is implicated in AD [BACE2 is associated with early AD, and its expression is negatively correlated with disease progression [BACE2 has been shown to be linked more to amyloid pathology than to neurofibrillary tangles pathology. This is supported by the evidence that it has a higher connectivity in the MTGnet, which is more susceptible to amyloid deposits than to neurofibrillary tangles [GLS2 ) was connected to 82 genes in the MTGnet and 17 genes in the PCCnet; 78 genes in the MTG and 3 genes in the HIP. Studies have shown large reductions in GLS2 activity in the brains of Alzheimer's subjects [GLS2 in the PCC and HIP, a further indicator of PCC and HIP showing signs of late AD pathology. Brain-specific tubulin polymerisation promoting protein (TPP/p25 ) had 60 neighbours in the MTGnet and 3 neighbours in the HIPnet. TPPP/p25 is an inhibitor of glycogen synthase kinase 3, thereby suppressing the phosphorylation of tau [PAK3 ) had 72 neighbours in the MTGnet and 3 in the HIPnet. Studies show that PAK3 levels were increased in early AD subjects, but declined in subjects with severe AD [We also examined the degree/connectivity of the zero TO genes in the coexpression networks . Furthermore, the same gene can have different functions depending on the physiological condition or brain region. Many genes have been known to play either destructive or protective roles depending on the condition, such as DNA repair genes allowing apoptosis or fighting against apoptosis. Furthermore, many genes also depict changes in their activity over the course of a disease. Our objective in this study was to analyse genes whose behaviour changed across regions, and determine if such changes in behaviour could characterise the difference in disease severity.union of the DE genes between two brain regions. In this case, some genes would be present in the network that were not considered differentially expressed in a certain region. Genes not considered significantly DE in any region could be due to 2 reasons - (a) noisy expression data and therefore not informative or (b) no change in expression in that region. We noticed that statistically significant DE genes with low gene expression tended to have lower network connectivity in that regional network. Moreover, if a gene has low expression in both regions or high expression in both regions, it does not have a large connectivity difference between the two regional networks. Since we are only analysing genes that have statistically significant differential expression, we can interpret downstream results with more confidence and not expect the results being due to chance. In our analyses on genes with a large difference in their network connectivity, we attribute this to their down regulation in a certain stage of AD because we know that certain regions get affected later on in the course of AD progression and therefore show early molecular changes occurring in AD. However, if genes that are not DE in a region are included in the network, we would not be able to ascertain whether the connectivity difference was due to statistically insignificant changes in expression  or because they were truly up or down regulated in that stage of AD.A natural step in this analyses could be the construction of gene co-expression networks using the There has not been much documentation of AD progression in the MTG , nor anyWe did not perform this study expecting the method to show that MTG would be less severely affected. After all the analyses of the different comparisons were completed, we were able to search for processes of interest  in the list of DE genes in the SFG and PVC as well as conclude that MTG is not severely affected. Nevertheless, by looking at the DE genes of the 4 AD affected regions one cannot clearly identify which processes to analyse further or reach such a conclusion. Since our conclusions regarding the MTG conform to what is already known from imaging and neuropathological reports, speaks well of the ability of this approach compared to other existing methods. Our results provide additional insights from a genetic and genomic perspective, bridging a gap between genetic information and disease phenotypes, and complement the findings from imaging and neuropathological studies. Furthermore, a conclusion from such analyses is a form of independent and unbiased conclusion. These facts pave the way for applying this novel method to investigate other lesser understood human diseases and conditions, as well as time course analysis, in order to generate testable hypotheses or, the more interesting scenario of comparing mild cognitive impairment (MCI) and AD. This paper has presented the application of this analysis method by applying to a well studied disease with a good microarray dataset so that results can be verified via literature search. Experimental validation of 100 s of genes is neither feasible nor cost effective. Therefore, it would be prudent to first test the method on well studied topics. We are currently applying this method (with good results) to study time-dependent and dose-dependent response to low-dose ionising radiation in a 3-D skin model (manuscript under preparation).We performed our analyses with the differentially expressed genes between AD affected and normal controls within each region for computational simplicity as well as to have more confidence in the interpretation of results as these DE genes are considered AD-perturbed genes. However, coexpression networks can be built using all the genes on the microarray chip. In order to preserve computational simplicity as well as include as many genes as possible, we used a relaxed FDR of 0.5% to select DE genes so that a larger number of genes could be common to the pairs of regions analysed.Although we proposed a particular measure of differential network topology, any valid measure of differential topology can be used. The same rationale applies to the TO threshold as well. The rationale is that network differences between data, that are more similar than different, may help in highlighting and understanding the causes of the differences in the data. From this study, it is postulated that methods relating to differential coexpression network topology should be included in the toolkit of techniques employed to study complex diseases such as Alzheimer's.The authors declare that they have no competing interests.MR and WZ conceived and designed the study. MR carried out the computational analysis and performed the biological interpretation. MR and WZ wrote the paper. All the authors read and approved the final manuscript.Number of network links for all regions. This excel sheet provides the number of network connection for all the genes in the network in each region.Click here for fileSix comparisons and twelve coexpression networks of the four regions. There were six comparisons among four brain regions. The coexpression networks for regions were built using the intersection genes of the regions being compared. The resulting networks built from the intersection genes are shown on the right.Click here for fileLists of TO genes. This excel sheet lists all the genes with zero topological overlap between regions.Click here for file


Intradermal vaccination provides direct and potentially more efficient access to the immune system via specialised dendritic cells and draining lymphatic vessels. We investigated the immunogenicity and safety during 3 successive years of different dosages of a trivalent, inactivated, split-virion vaccine against seasonal influenza given intradermally using a microinjection system compared with an intramuscular control vaccine.In a randomised, partially blinded, controlled study, healthy volunteers (1150 aged 18 to 57 years at enrolment) received three annual vaccinations of intradermal or intramuscular vaccine. In Year 1, subjects were randomised to one of three groups: 3 μg or 6 μg haemagglutinin/strain/dose of inactivated influenza vaccine intradermally, or a licensed inactivated influenza vaccine intramuscularly containing 15 μg/strain/dose. In Year 2 subjects were randomised again to one of two groups: 9 μg/strain/dose intradermally or 15 μg intramuscularly. In Year 3 subjects were randomised a third time to one of two groups: 9 μg intradermally or 15 μg intramuscularly. Randomisation lists in Year 1 were stratified for site. Randomisation lists in Years 2 and 3 were stratified for site and by vaccine received in previous years to ensure the inclusion of a comparable number of subjects in a vaccine group at each centre each year. Immunogenicity was assessed 21 days after each vaccination. Safety was assessed throughout the study.In Years 2 and 3, 9 μg intradermal was comparably immunogenic to 15 μg intramuscular for all strains, and both vaccines met European requirements for annual licensing of influenza vaccines. The 3 μg and 6 μg intradermal formulations were less immunogenic than intramuscular 15 μg. Safety of the intradermal and intramuscular vaccinations was comparable in each year of the study. Injection site erythema and swelling was more common with the intradermal route.An influenza vaccine with 9 μg of haemagglutinin/strain given using an intradermal microinjection system showed comparable immunogenic and safety profiles to a licensed intramuscular vaccine, and presents a promising alternative to intramuscular vaccination for influenza for adults younger than 60 years.Clinicaltrials.gov NCT00703651. Annual influenza epidemics cause substantial morbidity and mortality in all segments of the population, although some groups, such as elderly adults and individuals of any age with certain chronic conditions are especially at risk ,2. AccorIn healthy adults younger than 60 or 65 years , influenza vaccination coverage remains low despite being the most cost-effective intervention against annual influenza infection ,8. The sA new microinjection system  has been developed specifically to provide a convenient and reliable ID vaccination that overcomes the technical difficulties associated with historical ID injection methods. This easy-to-use system features a prefilled, ready-to-use syringe with micro-needle that protrudes 1.5 mm from the proximal end of the glass syringe . It has We report a trial conducted to assess the immunogenicity and safety of a trivalent, inactivated, split-virion influenza vaccine given intradermally using the new microinjection system. Several ID dosages were evaluated in comparison with a licensed inactivated influenza vaccine given intramuscularly. The trial lasted 3 years to investigate the safety of annual revaccination with the ID vaccine and annual vaccination alternating between ID and IM routes.The study was conducted in five centres in Belgium, one in the Czech Republic (Years 1 and 2 only) and three centres in Lithuania between September 2003 and May 2006. It was approved by the ethics committee of each centre before any patients were enrolled at that centre, and was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. All subjects gave written informed consent before entering the study.Healthy volunteers aged 18 to 57 years  were eligible. Exclusion criteria were: allergies to egg or chicken proteins or any of the vaccine constituents; a chronic illness in the active phase; acute febrile disease within 72 hours of any of the vaccinations or an axillary temperature >37.5°C on the day of vaccination; influenza vaccination within 6 months of the first study vaccination; any vaccination within 28 days of each study vaccination; pregnancy or breastfeeding; treatment with immunosuppressive or cancer therapy within 1 month of each vaccination, or an immunoglobulin injection within 3 months of each vaccination.This was a phase II, multicentre, randomised, partially blinded, dose-ranging study conducted over 3 successive years to assess the immunogenicity and safety of three annual vaccinations of ID, trivalent, inactivated, split-virion influenza vaccine in comparison with a licensed IM control vaccine. Each year, vaccination of all subjects occurred over a period of approximately 2 months (September and October). The primary objective of the study was to demonstrate, after vaccination in the first year of the study, that an ID vaccination of 3 μg or 6 μg of haemagglutinin (HA) per vaccine strain induced a non-inferior immune response compared with the IM control vaccine for all three vaccine strains. Secondary objectives included: assessment of compliance with the immunogenicity criteria defined in the European Medicines Evaluation Agency (EMEA) Note for Guidance , descripIn Year 1, 1150 subjects were randomised equally into three groups and received a first injection of either 3 μg or 6 μg of HA/strain intradermally or the IM control vaccine (15 μg HA/strain). The initial plan was to select either the 3 μg or 6 μg dosage based on the immunogenicity results from this first year to proceed with in Years 2 and 3. Subjects were to be randomised again in Year 2 and a third time in Year 3 into two equal groups and receive either the chosen ID dosage or the IM control vaccine. Statistical analysis performed with immunogenicity data collected after Year 1 vaccination showed that neither the 3 μg nor the 6 μg ID vaccine met the criteria for non-inferiority to the control IM vaccine. The protocol was therefore amended to continue vaccination in Years 2 and 3 with an escalated ID dose of 9 μg of HA/strain. The rationale for this amendment was two-fold: firstly to obtain descriptive immunogenicity data in a large sample size with this 9 μg ID formation before performing a formal assessment of non-inferiority versus the IM control in a follow-up trial, and secondly to assess the effect on safety of three annual ID vaccinations or alternating ID and IM vaccinations from year to year, as initially planned. This report will concentrate on the 9 μg vaccine formulation.Randomisation was performed using a permuted block randomisation method with decreasing block size. In Year 1, randomisation was stratified by centre. In Year 2, randomisation was stratified by centre and by vaccine actually received by each subject in Year 1. In Year 3, randomisation was again stratified by centre and by vaccine received actually received in Years 1 and 2. This randomisation strategy ensured that, each year, a comparable number of subjects were included in each vaccine group with an equal distribution of subjects vaccinated intradermally or intramuscularly in previous years  were trivalent inactivated, split-virion influenza vaccines . The antigenic dosage and strain composition of the investigational ID vaccines and the IM control vaccine given each year is shown in Table Both the investigational ID vaccines and the IM control vaccine (VaxigripThe primary endpoint was the geometric mean titre (GMT) of anti-HA antibodies for each of the three influenza strains 21 days after the first vaccination in each group. Antibody titres were determined in duplicate simultaneously for each strain before and 21 days after vaccination by the HA inhibition assay using the strains included in the vaccine each year  and are The following solicited reactions based on the EMEA Note for Guidance , occurriAssuming a standard deviation of 0.67 for the difference in log-transformed GMTs, it was calculated that 344 subjects per group were necessary to show non-inferiority  with a global power of 80.4%  and a significance level of 5%. To allow for 10% of subjects not being evaluable, 1146 subjects were to be included, 382 in each group.All statistical analyses were performed using SAS software, version 8.2 . The primary endpoint was analysed to determine whether the ID vaccine was non-inferior to IM vaccination. For both ID vaccine doses used in the first year, the ratio of GMTs after the first vaccination (ID/IM) and their 95% confidence intervals (CI) were calculated for each strain. To conclude non-inferiority of the ID vaccine, the lower bound of the 95% CI had to be greater than 1/1.5 for each strain. This analysis was performed, as planned in the protocol, on data collected from all study participants after the first vaccination and was performed with the aim of selecting one of the two ID vaccine formulations for further analysis in Years 2 and 3. If both the 3 μg and 6 μg ID vaccines had been found to be non-inferior to the IM control, then the lower of the two would have been selected. The ratio of GMTs (ID/IM) and their 95% CIs were also calculated to compare the immunogenicity of the ID 9 μg vaccine with the IM 15 μg vaccine after the second vaccination. 95% CIs were calculated for all immunogenicity outcomes using the normal approximate method for GMTs and GMTRs and using the Clopper-Pearson method for single percentages . Safety The primary analysis in Year 1 included all subjects who had conformed with the protocol and according to vaccine group allocation . Immunogenicity analysis after each vaccination was carried out on all subjects who had received all vaccinations up to that point and for whom pre- and post-vaccination titres were available. Safety analysis was performed on all available data.Of the 1150 subjects enrolled and randomised, 1149 were vaccinated in Year 1, 1091 were re-randomised and vaccinated in Year 2, and 828 were re-randomised and vaccinated in Year 3 . Four subjects died during the study; cause of death hepatocellular carcinoma, myosarcoma of the left thigh adductor, cerebral haemorrhage and myocardial infarction (98 other subjects experienced an SAE during the 3 years of the study). None were related to vaccination.Groups were equally matched in terms of both age and sex ratio; more women than men were enrolled Table .The 6 μg ID vaccination induced an immune response that satisfied the EMEA immunogenicity criteria  Table . HoweverAfter the second vaccination in Year 2, the immune responses against all strains were comparable with the 9 μg ID and control vaccine and all EMEA criteria were met for all strains in both groups Table . For eacAfter vaccination in Years 1, 2 and 3, the incidence of EMEA-specified reactions, most commonly shivering and malaise, was comparable between groups Table . ConsideInjection site pain and bruising occurred at a comparable rate in each group after vaccination with 9 μg ID or the control vaccine in Years 2 and 3 Table . Other sThe primary aim of this study was to identify the HA dose that, when administered intradermally using a microinjection system, would elicit an immune response that is statistically non-inferior to that elicited by a standard IM vaccination of 15 μg of HA/strain. In line with previous observations ,20,26, I®) as in our study.Despite the lower antigen content, the ID 9 μg vaccine was comparably immunogenic to the reference IM vaccine, satisfying EMEA criteria for all three virus strains. Although this study did not compare the same dosage given by ID and IM routes, these results are consistent with previous studies with rabies, hepatitis B and influenza vaccines that support the higher immunogenicity of the ID vaccination route -17. In aReactogenicity of the ID vaccine was comparable to that of the IM vaccine in terms of both EMEA reactions and solicited systemic reactions. It should be noted that the reactions listed in the EMEA Note for Guidance were specifically designed to determine the reactogenicity of IM vaccines and, as such, may not be fully appropriate for assessing reactogenicity after ID vaccination . As woulIt has been proposed that delivery of antigen via the ID route and associated activation of dermal dendritic cells favours the induction of a Th1-type cellular immune response, which can lead to delayed-type hypersensitivity ,29. RepeDespite the documented health and economic burden of influenza disease in adults younger than 60 years -34, surv® and IDflu®.As the primary outcome was not met in the first year of the study, our findings are limited by the fact that the immunogenicity of the 9 μg ID vaccine was descriptively compared with the IM vaccine after the second and third vaccinations. A formal statistical comparison of the 9 μg ID vaccine dose to the IM vaccine was not possible, as the population was not representative of an ID vaccination-naïve population. This formal comparison was done in a second trial that has been reported separately . These tWe have shown that a reduced dose, 9 μg/strain, of trivalent, inactivated, split-virion, seasonal influenza vaccine in a lower injection volume given intradermally using a novel microinjection system is as immunogenic as conventional IM vaccine with a comparable safety profile. Furthermore, our results show that the ID vaccine can be re-administered or interchanged with the IM vaccine annually without adversely affecting the safety profile. This vaccine administered using microinjection presents a promising alternative to IM vaccine for the vaccination of adults younger than 60 years against seasonal influenza.CI: confidence interval; EMEA: European Medicines Evaluation Agency; GMT: geometric mean titre; GMTR: geometric mean titre ratio; HA: haemagglutinin; ID: intradermal; IM: intramuscular; SAE: serious adverse event.FW and CS are employees of Sanofi Pasteur, the sponsor of this study. All other authors have no competing interests.The trial investigators JB, AA, AL, NM, PB, YC, ED, SH and PVB enrolled and followed subjects, revised and approved the final manuscript for publication. FW conceived the study, participated in its design, helped draft the manuscript and approved the final manuscript for publication, and was also the sponsor's responsible medical officer for the study. CS participated in the statistical design of the study, performed the statistical analysis and approved the final manuscript for publication.The pre-publication history for this paper can be accessed here:


In 2009, the new H1N1 pandemic virus exhibited several features that distinguished it from seasonal influenza: it caused major outbreaks in the northern hemisphere summer and autumn, it quickly dominated over other influenza viruses circulating in humans, and it caused widespread disease because of the lack of significant population immunity, particularly in young people. In 2010, the pandemic virus is behaving more like a seasonal influenza virus in that summer outbreaks have not been seen, it is co-circulating with seasonal A(H3N2) and B viruses, and the intensity of transmission is now lower than in 2009. For these reasons, the World Health Organization (WHO) downgraded its pandemic alert from phase 6 to the post-pandemic phase on 10 August 2010. Fortunately, in contrast to descriptions of the 1918 Spanish influenza pandemic, there has been no apparent change in disease severity over the first 18 months of circulation of this virus.Not necessarily, not altogether. Several features of this virus are a continued cause for concern; for example, most hospitalizations and deaths are still in those under 60 years old. This is probably because people in this age group are less likely to be immune. Furthermore, of those people admitted to hospital in the USA with confirmed influenza H1N1) 2009 pneumonia, almost two-thirds end up in intensive care. Recent clinical studies have identified risk factors for severe disease that include, but are not limited to, obesity, cardiovascular disease and pregnancy. Importantly, however, about one-third of those who have died with (H1N1) 2009 lacked any known risk factors N1 2009 p.  virus can replicate more extensively in the lower lung. Clinical data have shown that key risk factors for more severe infection include obesity, diabetes and immunosuppression among other underlying conditions [N1 virus The efficiency of influenza A (H1N1) 2009 virus transmission does not appear to be any greater than that of seasonal influenza. A key factor in the rapid and sustained global spread of the virus during 2009 was the very large pool of susceptible individuals due to low population immunity.But there have been some recent advances in identifying the molecular determinants of transmission - that is to say, the molecular factors that promote spread of the virus between individuals. Using influenza reverse genetics, two groups introduced known virulence determinants into the influenza A (H1N1) 2009 pandemic virus and used these viruses to study the impact of transmission in a ferret model of infection. Lysine at position 627 of the PB2 protein has been reported to be a virulence determinant in the highly pathogenic HPAI H5N1 avian influenza virus  and is aAnother factor is how well the virus binds to receptors in the airways. There is clear evidence that specific amino acids in the hemagglutinin molecule, particularly within the binding site whereby it recognizes its receptor on cells, dictate specificity for either α2,3- or α2,6-linked sialic acids. Human influenza viruses have an aspartic acid (D) at positions 190 and 222 in the hemagglutinin that impart α2,6-sialic acid binding. In contrast, the avian influenza virus preferentially recognizes α2,3-linked sialic acids, and this preference is determined by glutamic acid (E) and glycine (G) at positions 190 and 222, respectively. Of particular interest was an experiment reported by Tumpey and colleagues at the recent Options for the Control of Influenza VII meeting in Hong Kong early in September. They used a mouse-adapted pandemic (H1N1) strain with a D to G mutation at position 222 of hemagglutinin . This waTwo neuraminidase inhibitors have been widely used in the prophylaxis or treatment of pandemic (H1N1) influenza: oseltamivir (marketed as Tamiflu) and, to a lesser extent, zanamivir (Relenza). Oseltamivir-resistant pandemic strains have been detected, often associated with prolonged treatment of severe cases, but to date there is little evidence of sustained spread of these strains among untreated individuals. As the most common oseltamivir resistance mutation (an H to Y change at position 275) is close to the substrate-binding site of the neuraminidase protein, it was expected from earlier animal studies that such mutants would be less transmissible than their wild-type counterparts. There are conflicting data on this issue. For example, the H275Y oseltamivir resistance mutation emerged in seasonal (H1N1) viruses in late 2007 and spread globally during 2008 in the absence of widespread usage of the drug, suggesting that the mutation had not impaired viral transmissibility. Subsequent work has identified 'permissive' mutations that restored the fitness of these viruses . There aInitial clinical trials demonstrated that the monovalent influenza (H1N1) 2009 vaccine is immunogenic and capable of inducing levels of antibody that are considered protective . VaccinaThat said, it may still be too early to tell just how effective vaccination against the pandemic virus has been. There are two reasons for this. The first is that the initial roll-out of the vaccine occurred too late to affect the first pandemic wave. For example, Australia received the monovalent vaccine in late September 2009. Although this was only 5 months after selection of the vaccine strain, winter was over and the initial pandemic wave had subsided. Importantly, there has been strong collective uptake of the monovalent pandemic vaccine and the later trivalent seasonal vaccine (which included the pandemic (H1N1) strain) in Australia, probably because of an effective public education program. According to recent Australian Government reports, influenza activity is rising even though spring is now beginning in Australia. It will be interesting to see whether the delay in onset of the influenza season and its relatively low activity is due to the extensive vaccination program. We have to wait and see.The most important impediment to vaccine intervention during the early stages of the pandemic was a delay in availability. Although the full sequence of the new virus was publicly available within days of its identification in April 2009 and a suitable vaccine strain was recommended by WHO just one month later, vaccine production and deployment were significantly delayed by low virus yields in eggs and a number of regulatory hurdles. As a consequence, there is a lot of interest in developing new vaccine strategies that generate more broadly cross-reactive immunity. More recent advances have been in generating antibody responses against conserved regions of the hemagglutinin protein rather than the more variable regions found within the globular head of the protein. In a recent report, Gary Nabel and colleagues demonstrated that a DNA/recombinant adenovirus prime-boost strategy generated antibodies that cross-reacted with antigenically distinct influenza strains . They weAlthough it took only a few days from initial identification of the influenza (H1N1) 2009 virus in Mexico and California in April to the announcement by WHO that its emergence was a public health event of international concern , the virus had in fact been circulating in humans for at least 2 months. Could we have acted earlier to prevent or reduce the impact of the pandemic? More specifically, if we had more active surveillance in the pig population, would this have accelerated detection and response to the new virus in humans? Yes, but not on its own. Early detection of new reassortants in pigs will be useful if it triggers enhanced surveillance for human cases. The most critical need is for rapid detection and laboratory investigation of unusual disease outbreaks combined with open sharing of information and material with national and international health authorities.Fortunately, our worst fears were not realized in the influenza A (H1N1) 2009 pandemic. However, despite all the advances in technology, surveillance and pandemic planning, the virus spread globally within months, reminding us how difficult it is to control. There were a number of positive outcomes. One was the rapid sharing of information and strains between different parties around the world. This was critical in helping governments and international agencies to shape an appropriate response to an uncertain threat and in enabling manufacturers to produce a new vaccine within 5 months of the first detection of the virus. While some quarters have criticized the response as excessive, it is likely the pandemic would have posed a greater problem in the absence of such interventions. Another positive outcome was the opportunity to evaluate the effectiveness of pandemic plans with a view to ensuring improvements. Furthermore, the emergence of pandemic influenza (H1N1) 2009 stimulated a large amount of research, resulting in new and important knowledge about the virus itself - all important for refining and strengthening our preparedness for future pandemics.