Rashida Ferrand and colleagues show that HIV infection is the commonest cause of hospitalization among adolescents in a high HIV prevalence setting. Survival to older childhood with untreated, vertically acquired HIV infection, which was previously considered extremely unusual, is increasingly well described. However, the overall impact on adolescent health in settings with high HIV seroprevalence has not previously been investigated.z-score<−2: 52% versus 23%, p<0.001), have pubertal delay , and be maternal orphans or have an HIV-infected mother . 69% of HIV-positive and 19% of HIV-negative admissions were for infections, most commonly tuberculosis and pneumonia. 84 (28%) participants had underlying heart, lung, or other chronic diseases. Case fatality rates were significantly higher for HIV-related admissions , and significantly associated with advanced HIV, pubertal immaturity, and chronic conditions.Adolescents (aged 10–18 y) systematically recruited from acute admissions to the two public hospitals in Harare, Zimbabwe, answered a questionnaire and underwent standard investigations including HIV testing, with consent. Pre-set case-definitions defined cause of admission and underlying chronic conditions. Participation was 94%. 139 (46%) of 301 participants were HIV-positive (median age of diagnosis 12 y: interquartile range [IQR] 11–14 y), median CD4 count = 151; IQR 57–328 cells/µl), but only four (1.3%) were herpes simplex virus-2 (HSV-2) positive. Age (median 13 y: IQR 11–16 y) and sex  did not differ by HIV status, but HIV-infected participants were significantly more likely to be stunted  has killed more than 25 million people since 1981, and more than 30 million people are now infected with the human immunodeficiency virus (HIV) that causes AIDS. HIV destroys the cells in the immune system that normally provide protection against disease-causing organisms. Consequently, people infected with HIV are susceptible to so-called opportunistic infections, including tuberculosis and pneumonia. HIV is most commonly spread through unprotected sex with an infected partner but another major route of transmission is mother-to-child  during pregnancy or delivery or during breast feeding. Mother-to-child transmission can be prevented by giving antiviral drugs to HIV-positive mothers during their pregnancy and to their newborn children. But, although most mothers in developed countries have access to this intervention, fewer than half of HIV-positive mothers in low- and middle-income countries receive this treatment and, every year, nearly half a million children become infected with HIV.It is generally thought that HIV infections in infants progress rapidly and that half of the children who acquire HIV from their mothers will die before their second birthday if not treated with antiretroviral drugs. However, as the AIDS epidemic matures, more children are surviving to adolescence with untreated, vertically acquired HIV infection in sub-Saharan Africa, the region where most children with HIV/AIDS live. Little is known about the burden of HIV infection and its contribution to illness and death in adolescents in sub-Saharan Africa but this information is needed to help health care providers prepare for this new aspect of the AIDS epidemic. In this study, the researchers examine the causes of acute hospital admissions  among adolescents in Zimbabwe, a country where the HIV epidemic started early and where one in seven adults is HIV-positive and more than 17,000 children are infected with HIV every year, mainly through vertical transmission.The researchers recruited 301 10–18-year olds who were admitted to each of the two public hospitals in Harare (Zimbabwe) for acute illnesses between September 2007 and April 2008. Each patient completed a questionnaire about themselves and their health and underwent standard investigations, including HIV testing. Nearly half the participants were HIV positive; about a quarter of these HIV-positive individuals only found out about their status during the study. HIV-positive participants were more likely to be stunted, to have pubertal delay, and to be maternal orphans or have an HIV-infected mother than HIV-negative participants. 69% of HIV-positive participants were admitted to hospital because of infections, often tuberculosis or pneumonia whereas only 19% of the HIV-negative participants were admitted for infections. More than a quarter of all the participants had underlying heart, lung, or other chronic conditions. Finally, 22% of the HIV-positive participants died while in hospital compared to only 7% of the HIV-negative participants. Factors that increased the risk of death among all the participants were advanced HIV infection, pubertal immaturity, and chronic conditions.These findings indicate that HIV infection is the commonest cause of acute adolescent admission to hospital in Harare (and probably elsewhere in Zimbabwe). Most of these admissions are due to opportunistic infections similar to those seen in HIV-positive adults and to long-term complications of having HIV/AIDS as an infant such as delayed puberty. Other findings indicate that most of the HIV-positive adolescents who participated in this study were infected via vertical transmission, which supports the idea that long-term survival after vertical infection is possible. Because the AIDS epidemic started early in Zimbabwe, there is likely to be a lag before adolescent survivors of vertical HIV transmission become common elsewhere. Nevertheless, all African countries and other places where HIV infection in adults is common need to recognize that the burden of HIV in their acutely unwell adolescents is likely to increase over the next few years. To deal with this emerging aspect of the AIDS epidemic, measures must be introduced to ensure early diagnosis of HIV in this previously neglected age group so that treatment can be started before HIV-positive adolescents become critically ill.http://dx.doi.org/10.1371/journal.pmed.1000178.Please access these Web sites via the online version of this summary at PLoS MedicinePerspective by Glenda GrayThis study is further discussed in a HIV infection and AIDSInformation is available from the US National Institute of Allergy and Infectious Diseases on HIV InSite has comprehensive information on all aspects of HIV/AIDS, including a list of articles and other sources of information about the primary care of adolescents with HIVAvert, an international AIDS charity on many aspects of HIV/AIDS, including information on the HIV and AIDS in Zimbabwe, and on children, HIV, and AIDS (in English and Spanish)Information is available from children and HIV and AIDS UNICEF also has information about  In industrialised countries trauma and behavioural disorders, such as substance abuse, obesity, and sexually transmitted infections account for most adolescent morbidity As the HIV epidemic matures, long-term survival of HIV-infected infants to adolescence following vertical transmission is increasingly being recognised in African countries The spectrum of HIV-related disease varies considerably between adults and children, but has not been well-defined for adolescents. Although opportunistic infections predominate in all age groups, chronic noninfectious clinical conditions may be more prominent in HIV-infected adolescents than in adults and infants Harare Central Hospital and Parirenyatwa Hospital are the main public sector hospitals in Harare and cater to two-thirds of Harare's population; the remaining one-third using private health care facilities. The cost of hospitalisation in public sector facilities is partially subsidised by the government. Malaria transmission does not occur in Harare. Between September 2007 and April 2008, patients admitted to either hospital were enrolled consecutively the following day. Recruitment was limited to weekdays and to a maximum of five patients per site per day for logistical ease. Patients aged between 10 and 18 y admitted with any acute medical or surgical condition, including trauma, were eligible. Patients were excluded if moribund , requiring intensive care admission, or admitted for obstetric or elective reasons, or previously enrolled into the study.Pneumocystis jirovecii infection was suspected, patients underwent sputum induction with nebulised hypertonic saline.Standardised investigations, summarised in Otherwise investigations followed standard hospital guidelines including lumbar puncture for suspected meningitis and cerebral computed tomography (CT) for focal neurological signs or suspected encephalopathy. Participants were followed for the duration of their hospital stay. Pre-set diagnostic algorithms defined the definitive or presumptive cause of admission, and any underlying chronic conditions . The AduHIV serology used parallel testing with Abbott Determine and SD Bioline. Discordant samples were resolved using Vironostika. HSV-2 antibodies were detected using recombinant IgG antigen . CD4 counts were determined by flow cytometry .Cryptococcus using India ink contrast staining, culture on Sabouraud media, and cryptococcal antigen testing . Induced sputum was stained with Grocott's silver stain. Thick blood films for malaria were examined using Giemsa staining.Blood culture used Myco/F Lytic  t-test for normally distributed variables and Mann Whitney U test for variables not normally distributed. Categorical variables were compared using the Chi-squared (χ2) or Fisher's exact test as appropriate. Z-scores for height- and weight-for-age were calculated using British 1990 Growth Reference Curves, which provide data over the age of 10 y z-Scores of <−2 were considered to represent stunting and wasting, respectively Data were analysed with STATA software, version 10.0 (STATA Corporation). Continuous variables were compared using Student's p<0.05 were included in a multivariate model. Factors that remained independently associated with death were retained. The association between each factor in the clinical group and death was assessed by adding each factor into the multivariate model that included the subset of independently significant socio-demographic factors. The multivariate model was built that included the subset of socio-demographic factors in the first multivariate model plus any clinical factors that were significant after adjusting for the socio-demographic factors. The final multivariate logistic regression model was reached by excluding single factors sequentially until all remaining factors were statistically significant.Multivariate logistic regression was used for analysis of risk factors for mortality Written consent was obtained from all participants and from guardians of participants aged below 16 y. Onward referral for HIV care services was made for all testing HIV-positive. Guardians were encouraged to disclose HIV status to HIV-infected participants who did not know their status. Ethical approval for the study protocol was obtained from the Ethics Committees of the London School of Hygiene and Tropical Medicine and the Joint Research Ethics Committee at the University of Zimbabwe, Medical Research Council of Zimbabwe (MRCZ), Harare and Parirenyatwa Hospital Ethics Committees and the Institutional Review Board of the Biomedical Research and Training Institute.p<0.032), although the comparison was based on small numbers.Of 1,025 total adolescent hospital admissions, 340 were eligible. 301 participants were recruited, with a refusal rate of 6% . HIV preThe median age at diagnosis of HIV infection was 12 y (IQR: 11–14). Of the 139 participants who were HIV-positive, 86 (62%) had tested prior to admission and knew their HIV status. Fifty participants tested positive following PITC; of these, 17 had tested HIV-positive previously but had not been told of their HIV infection. All guardians, however, agreed to disclosure to the participant with assistance of study counsellors. Only 18 (6%) participants declined PITC, of whom three were HIV-positive on unreported HIV testing and are likely to remain unaware of their HIV infection.HIV-infected participants were significantly more likely to be orphans, previously treated for tuberculosis, previously hospitalised more than once, stunted, and pubertally immature  .HIV-infected patients were profoundly immunosuppressed at presentation: 83% had WHO stage 3 or 4 disease and 58% had CD4+ T lymphocyte counts <200 cells/µl. Only 44 (43%) of the HIV-infected participants who had previously had an HIV test were on ART (for a median duration of 121 d).The most frequent diagnosis among HIV-infected participants was infection with tuberculosis, pneumonia, cryptococcosis, and blood stream infections being the most frequent diagnoses. Among HIV-negative participants, the commonest cause of admission was trauma, followed by acute exacerbations of chronic medical conditions, predominantly cardiac . The medThe highest median CD4 counts were found in patients presenting with trauma or acute surgical causes conditions (455 and 628 cells/µl). Patients with blood stream infections, oesophageal candidiasis, and HIV-wasting syndrome had the lowest median CD4 counts .p<0.001) met criteria for the standardised infectious screen  culture in five patients, and seven patients had a positive cryptococcal antigen (CrAg) only.113 (81%) and 41 (25%) of HIV-infected and HIV-negative admissions . p<0.56). In addition, 70% of HIV-infected participants had chronic skin complaints, although rarely responsible for admission (p<0.082). Chronic lung disease and cardiac disease were the most common serious HIV-related complications (84 (28%) participants had underlying chronic medical conditions other than HIV . Death was significantly associated with low CD4+ T lymphocyte count . The highest case-fatality rates among HIV-infected participants were from HIV wasting syndrome (53%), any malignancy (50%), and drug toxicity (50%), and among HIV-negative participants the highest case-fatality rates were for malignancy (83%) (32/139 (23%) HIV-infected participants died in hospital compared with 11/162 (7%) HIV-negative participants  .p<0.032), chronic disease , pubertal delay , and poor performance scores  remained independently associated with increased risk of death.Of the considered socio-demographic risk factors, younger age and having a primary care-giver who was not the parent were associated with an increased risk of death . After aThe main finding of this study is that HIV is now the single most common cause of acute admission and in-hospital death among adolescents in Harare. HIV-infected adolescents were profoundly immunosuppressed, and the median CD4 count (51 cells/µl) was similar to that reported in other studies of hospitalised African adults in the pre-ART era We found an equal sex distribution of HIV infection, low rates of self-reported sexual debut, and a much lower prevalence of HSV-2 infection than would be anticipated for sexually acquired HIV in southern Africans Diagnosis of HIV infection was frequently delayed in this age group until presentation with advanced HIV disease. A substantial minority had not been diagnosed with HIV before hospitalisation, and most others reported relatively recent diagnosis following a prolonged history of recurrent infections. The beneficial effects of early diagnosis and ART on reducing the risk of opportunistic infections and chronic complications, reducing mortality, and improving growth are well-recognised in children Although the phenomenon of long-term survival in HIV-infected infants is well recognised in Zimbabwe, several factors unique to this age group may be creating both demand and supply-side barriers to diagnostic testing: in contrast to adults, there are no free-standing counselling and testing services for under 16-y-olds in Zimbabwe, and there are also legal barriers to testing without the permission of the legal guardian, who may be incapacitated or absent. Reluctance of guardians to disclose the true nature of the underlying illness was also apparent in that a substantial minority of our HIV-infected participants had not been told their previous test results. Clear advice to health professionals to offer PITC and to assist guardians with disclosure may improve timely diagnosis and adherence to subsequent ART Our study was hospital-based and therefore likely to be biased towards sicker patients and more severe HIV-associated complications. One of the study sites was based at a referral hospital, which may have resulted in a higher proportion of specialist diagnoses such as cancer. Both hospitals had ART clinics, but only nine (6%) of the HIV-infected participants were admitted from these. The vast majority of our participants (87%) were referred directly from primary health care clinics or through hospital casualty departments, and the only local alternatives to these two hospitals are private facilities. Our results are, therefore, likely to be representative of the pattern of acute severe morbidity and mortality in Harare. Judging by the high numbers of adolescents attending HIV care clinics throughout Zimbabwe HIV-related morbidity and mortality, most likely reflecting long-term survival from the paediatric HIV epidemic in the 1990s, is now a major cause of adolescent morbidity and mortality in Harare. Recognition of the high burden of HIV in acutely unwell older children and adolescents is needed to stimulate earlier diagnosis and improve access to HIV care for this previously neglected age group. Our results strongly support implementation of PITC for all patients in this age group attending health facilities, ideally at the primary care as well as hospital level, so that diagnosis is made and treatment can be started before patients are critically ill.Text S1Case definitions used during the present study.(0.04 MB DOC)Click here for additional data file.


Infections with hepatitis C virus (HCV) and, possibly, hepatitis B virus (HBV) are associated with an increased risk of non-Hodgkin's lymphoma (NHL) in the general population, but little information is available on the relationship between hepatitis viruses and NHL among people with HIV (PHIV). We conducted a matched case–control study nested in the Swiss HIV Cohort Study (SHCS). Two hundred and ninety-eight NHL cases and 889 control subjects were matched by SHCS centre, gender, age group, CD4+ count at enrolment, and length of follow-up. Odds ratios (OR) and corresponding 95% confidence intervals (CI) were computed using logistic regression to evaluate the association between NHL and seropositivity for antibodies against HCV (anti-HCV) and hepatitis B core antigen (anti-HBc), and for hepatitis B surface antigen (HBsAg). Anti-HCV was not associated with increased NHL risk overall , or in different strata of CD4+ count, age or gender. Only among men having sex with men was an association with anti-HCV found . No relationships between NHL risk and anti-HBc or HBsAg emerged. Coinfection with HIV and HCV or HBV did not increase NHL risk compared to HIV alone in the SHCS. People infected with HIV (PHIV) have an approximately 100-fold increased risk of non-Hodgkin's lymphoma (NHL), mainly high-grade B-cell NHL, compared to the general population . The rolAn association between hepatitis C virus (HCV) infection and NHL in the general population has emerged in the last decade .Owing to shared routes of transmission, HCV and HIV coinfection is common and is associated with higher HCV RNA levels and increased risk of progression of HCV-related liver disease than in the presence of HCV infection alone  to assess the relationship between HCV infection and NHL in PHIV. On account of some reports of increased NHL risk in HIV-negative individuals seropositive for hepatitis B surface antigen (HBsAg) (www.shcs.ch). Follow-up visits take place every 6 months and clinical events, such as opportunistic diseases, selected cancers , and death have always been recorded.The SHCS is an ongoing study that has been enrolling PHIV since 1988, with some retrospective enrolment going back to 1984, from seven large hospitals in Switzerland  at enrolment. For five control subjects, the retrieved blood sample was inadequate for anti-HCV testing, leaving 889 control subjects available for the present study  centre of enrolment in the SHCS; (2) gender; (3) age group (in 5-year groups from 16 to 20 to ⩾65 years); and (4) CD4+ count  than for control subjects . Median year at cancer diagnosis for NHL cases was 1995 (interquartile range: 1993–1998).HIV-transmission category was classified into three groups: (1) intravenous drug users (IDUs), (2) men having sex with men (MSM), and (3) heterosexual and others. ‘Others’ (i.e. transmission through blood and blood products and unknown transmission category) included only 14 NHL cases and 36 control subjects.This study was approved by the local ethical committees of the clinics collaborating with the SHCS and of the International Agency for Research on Cancer. Written informed consent was obtained from all SHCS participants.All hepatitis virus markers were measured in blood samples taken before NHL diagnosis or the corresponding reference date for controls. As systematic anti-HCV testing in the SHCS started in April 1998, anti-HCV results could not be obtained from medical records for 172 (57.7%) NHL cases and 261 (29.4%) control subjects. New HCV testing on stored serum aliquots was, therefore, performed at the Institute for Medical Microbiology, University of Basel, Switzerland using third–generation ELISA . Reactive results were confirmed by immunoblot. Among 51 PHIV for whom anti-HCV results were available from both medical records and new HCV testing, anti-HCV status agreed in 50.Information on antibodies against hepatitis B core antigen (anti-HBc) came from SHCS records for 100 NHL cases and 539 control subjects. New testing was performed on stored serum aliquots from 197 NHL cases and 347 control subjects, using microparticle enzyme immunoassay . Anti-HBc status could not be evaluated in one NHL case and three controls whose anti-HCV status was known.For HBsAg, information came from SHCS records for 82 NHL cases and 472 control subjects. New testing was performed on stored serum aliquots from 210 NHL cases and 371 control subjects, using microparticle immunoenzyme assay . Hepatitis B surface antigen status could not be evaluated in six NHL cases and 46 controls whose anti-HCV status was known.Testing for antibodies against HBsAg and against HBe antigen was not systematically carried out in the SHCS, and testing for HCV RNA started only in February 2002. Thus, these three additional viral markers were not considered in the present study. The CD4+ count was measured by flow cytometry.Conditional logistic regression was used to calculate odds ratios (ORs) and corresponding 95% CIs. All analyses were conditioned on all matching variables (see Subjects) and adjusted for HIV-transmission category and year of enrolment . Additional adjustment for highly active antiretroviral therapy (HAART) use did not substantially change the ORs reported. Unconditional logistic regression was used for the analyses stratified by HIV transmission category, adjusted for all matching variables and year of enrolment.χ2 distribution with degrees of freedom equal to the number of strata minus one . In no instance, however, was the effect of anti-HCV seropositivity on NHL risk significantly heterogeneous across the strata of the variables considered .+ and anti-HCV− NHL cases. Although 95% CIs always overlapped, a slightly lower proportion of anti-HCV+ than anti-HCV− NHL cases had less than 50 CD4+ cells μl−1 at cancer diagnosis , or were diagnosed with PBL .Seropositivity for HBV and HCV did not increase NHL risk among PHIV in the SHCS. Our findings on HCV, the most studied hepatitis virus in respect to NHL risk , are conAs expected, levels of seropositivity for hepatitis viruses were high among PHIV, most notably among IDUs. The higher anti-HBc/anti-HCV seropositivity ratio among MSM (16.7) and heterosexuals and other (3.4) compared to IDUs (0.90) confirms the much greater efficiency of the sexual-route of transmission for HBV than HCV .+ and anti-HCV− lymphomas. We did detect, however, a slight under-representation of anti-HCV+ compared to anti-HCV− cases among NHL occurring at CD4+ counts below 50 cells μl−1 and among PBL, which are the two lymphoma subtypes where EBV is known to be most strongly implicated  were older than subjects in the other HIV-transmission categories . A two-fold increased NHL risk was also found among anti-HCV+ PHIV aged 45 years or older, although it did not reach statistical significance. Older age may be a correlate of longer exposure to HCV, and, hence, higher risk of HCV-related complications including NHL. Indeed, the relative risk for cirrhosis in anti-HCV+vs anti-HCV− PHIV increased between the pre-HAART and HAART era . Weaknesses of the SHCS include incomplete information on time of HIV seroconversion, which prevented us from using years of follow-up as an exact proxy for duration of HIV infection. In addition, no information was available on EBV infection and on the activity of HCV infection, that is the presence of serum HCV RNA. The vast majority of persons acutely infected with HCV develop chronic persistent viraemia and, especially among PHIV, anti-HCV seropositivity is likely to correspond to chronic HCV infection (An important strength of the SHCS is the fact that it is very representative of PHIV in Switzerland. It has been estimated that, since the beginning of the HIV epidemic, 48% of PHIV, and 68% of people diagnosed with AIDS in Switzerland have been enrolled in the SHCS (In conclusion, coinfection with HIV and HCV or HBV did not increase NHL risk compared to HIV alone in the SHCS. However, as the life expectancy of PHIV increases, the influence of HCV infection on cancer risk deserves further study as the high prevalence of HCV could result in huge attributable risks, even in the presence of weak relative risks.


A method of carrying out region-wide audit for breast cancer was developed by collaboration between the cancer registry, providers and purchasers as part of work to fulfill the 'Calman-Hine' recommendations. In order to test the audit method, a retrospective audit in North Thames East compared practice in 1992 against current guidelines. The analysis compared care in specialist and non-specialist centres. A stratified random sample comprising 28% of all breast cancer patients diagnosed in 1992 was selected from the population-based Thames Cancer Registry. The data for 309 patients with stage I-III tumours were analysed by hospital type using local guidelines. No difference between specialist (high volume) and non-specialist centres was detected for factors important in survival. Pathological staging was good with over 70% reporting tumour size and grade. A small number of patients were undertreated; after conservative surgery, 10% (19) of women did not receive radiotherapy, and 15% (8) of node-positive premenopausal women did not receive chemotherapy or ovarian ablation. In contrast, a significant trend with hospital volume was found for several quality of life factors. These included access to a specialist breast surgeon and specialist breast nurses, availability of fine-needle aspiration (FNA), which ranged from 84% in high-volume to 42% in low-volume centres, and quality of surgery (axillary clearance rates ranged from 51% to 8% and sampling of less than three nodes from 3% to 25% for high- and very low-volume centres respectively). Confidential feedback of results to surgeons was welcomed and initiated change. The summary information gave purchasers information relevant to the evaluation of cancer services. While the audit applied present standards to past practice, it provided the impetus for prospective audit of current practice (now being implemented in North Thames).


Initiation and regulation of immune responses in humans involves recognition of peptides presented by human leukocyte antigen class II (HLA-II) molecules. These peptides (HLA-II T-cell epitopes) are increasingly important as research targets for the development of vaccines and immunotherapies. HLA-II peptide binding studies involve multiple overlapping peptides spanning individual antigens, as well as complete viral proteomes. Antigen variation in pathogens and tumor antigens, and extensive polymorphism of HLA molecules increase the number of targets for screening studies. Experimental screening methods are expensive and time consuming and reagents are not readily available for many of the HLA class II molecules. Computational prediction methods complement experimental studies, minimize the number of validation experiments, and significantly speed up the epitope mapping process. We collected test data from four independent studies that involved 721 peptide binding assays. Full overlapping studies of four antigens identified binding affinity of 103 peptides to seven common HLA-DR molecules . We used these data to analyze performance of 21 HLA-II binding prediction servers accessible through the WWW.ROC), 17 predictors showed good (AROC > 0.8), 41 showed marginal (AROC > 0.7), and 55 showed poor performance (AROC < 0.7). Good performance predictors included HLA-DRB1*0101 (seven), 1101 (six), 0401 (three), and 0701 (one). The best individual predictor was NETMHCIIPAN, closely followed by PROPRED, IEDB (Consensus), and MULTIPRED (SVM). None of the individual predictors was shown to be suitable for prediction of promiscuous peptides. Current predictive capabilities allow prediction of only 50% of actual T-cell epitopes using practical thresholds.Because not all servers have predictors for all tested HLA-II molecules, we assessed a total of 113 predictors. The length of test peptides ranged from 15 to 19 amino acids. We tried three prediction strategies – the best 9-mer within the longer peptide, the average of best three 9-mer predictions, and the average of all 9-mer predictions within the longer peptide. The best strategy was the identification of a single best 9-mer within the longer peptide. Overall, measured by the receiver operating characteristic method (AThe available HLA-II servers do not match prediction capabilities of HLA-I predictors. Currently available HLA-II prediction servers offer only a limited prediction accuracy and the development of improved predictors is needed for large-scale studies, such as proteome-wide epitope mapping. The requirements for accuracy of HLA-II binding predictions are stringent because of the substantial effect of false positives. Vaccines are the most effective means for fighting against infectious diseases . They arThe peptide-binding groove of a MHC molecule consists of a β-sheet and two α-helices. A peptide binds through a network of hydrogen bonds between its backbone and the binding groove, and through interactions between the peptide side chains and pockets inside the binding groove ,7. Most The ability of the immune system to respond to a particular antigen differs between individuals because they display different patterns of MHC genes. Human MHC molecules are known as human leukocyte antigens. Each human individual expresses up to six HLA-I molecules and up to a dozen HLA-II molecules. HLA genes show extensive polymorphism. As of August 2008, more than 3000 HLA alleles have been identified and sequenced including 2215 HLA-I and 986 HLA-II sequences . The divIdentification of HLA binding molecules is, therefore, important for both understanding the basing molecular function of the immune system and for vaccine development. However, systematic T-cell epitope mapping is costly and time-consuming because it involves synthesis and testing of overlapping peptides spanning the full length of target antigens. For short peptides such as tumor antigen surviving (BIRC5), that is 142 amino acids long, full overlapping studies of both HLA-I and -II binders were performed for several HLA molecules ,14. HoweComputational prediction of peptide binding to MHC molecules has been a topic of vigorous research and development activity -22. Comp• Only a small fraction of peptides in a given pathogen or tumor-specific proteome are able to bind to a specific MHC molecule . Tens of• The comparison studies performed to date have been based on assessing predictive performance using pre-defined sets of peptides, rather than well-defined standardized full-overlapping studies of complete antigens. This introduces biases and the reported performances are likely to be overestimates.• HLA-II peptide binding is mediated through 9-mer binding core, but longer peptides are used for experimental measurement of binding. Hereby we predict one element (the 9-mer binding cores) and experimentally test with another element . This makes the improvement of false positive rate an important issue in prediction of HLA-II binding and it requires sophisticated statistical and machine learning approaches see ,29,34).,34.29,34• Both ends of the peptide binding grooves in HLA-II molecules are open, allowing the peptides to be more variable in length  and flanking residues are known to selectively affect binding . This ef• Some longer peptides bind MHC-II through multiple overlapping 9-mer registers ,35 addin• Experimental measurements of HLA-II binding shows variation depending on the conditions of the experiment, even for the control peptides.• Sufficient quantities of HLA-II binding data are available only for some HLA-DR molecules while, notwithstanding notable exceptions , HLA-DQ • Presentation of HLA-II binding peptides depends on antigen processing steps including editing by HLA-DM and other accessory molecules. DM editing affects the density and preference for particular peptide species . These eHLA-II binding predictions are thus more complex than HLA-I predictions ,37,38. VThis study extends the assessment of predictive power to include a much larger number of servers that predict HLA-II binding. This study was limited to seven common HLA-DR molecules that have sufficient amount and quality of peptide binding data. We compiled and established standardized test data sets that are more representative of the experimental reality, and defined a uniform scaling scheme to use data from different studies. Finally we assessed the practical applicability of HLA-II binding predictions to identification of HLA-II T-cell epitopes. Our study identified several key issues that need to be addressed for the development of improved prediction systems of HLA-II binding.While not all the servers were designed specifically for peptide binding predictions, all of them have implemented modules for this step. Some servers also have advanced options, for example, MHCPred enables users to specify anchor positions. For this analysis we used the simplest prediction method available at each server. The numbers of the servers for individual HLA-DR alleles we studied were: HLA-DRB1*0101 – 19, HLA-DRB1*0301 – 15, HLA-DRB1*0401 – 20, HLA-DRB1*0701 – 16, HLA-DRB1*1101 – 17, HLA-DRB1*1301 – 9, and HLA-DRB1*1501 – 17.ROC values of these predictions are shown in Figure In total 113 individual predictors were tested of which 17 showed good, 41 marginal, and 55 poor performance using the single maximum 9-mer prediction scheme. 8 showed good, 30 marginal, and 75 poor performance using the average prediction for all 9-mers within the test peptide. Using the average of best of three 9-mer predictions, 12 servers showed good, 37 marginal, and 64 poor, performance. The AROC = 0.9 while other "good" predictors are close to the lower borderline leaving ample space for the improvement.Comparing the prediction performance across HLA-DR alleles, the best predictors are for HLA-DRB1*0101, where seven predictors showed good classification accuracy, while six DRB1*1101 predictors, three DRB1*0401 predictors, and only one DRB1*0701 predictor showed good classification accuracy. None of predictors for DRB1*0301, DRB1*1301, and DRB1*1501 showed good classification performance. Important to note, only four HLA-DRB1*0101 predictors have shown performance that approaches the value of AThe best prediction server across all HLA molecules evaluated in this study is NETMHCIIPAN, closely followed by PROPRED, IEDB_SAT, and MULTI_SVM. The best predictors we recommend for each allele are marked by asterisks in Figure ROC was then calculated and the results are shown in Figure ROC values higher than 0.775. DR4_ANN and DR4_SVM predictors were excluded from this analysis since they predict peptide binding to single MHC-II allele (HLA-DRB1*0401). To enable the comparison of predictions that include multiple HLA alleles, we developed a common scaling scheme for seven HLA-DRB1 alleles.  Binding scores used in this scheme range from 0 to 100 and threshold for binding is at 50. The scaled data are accessible at DFRMLI [Promiscuous peptides are able to bind to multiple MHC molecules. Therefore they serve as promising targets for vaccine design because they are likely to cover a larger population of patients . We perft DFRMLI .We also assessed the performance of prediction servers in identification of tumor antigen T-cell epitopes. For each server we predicted the binding affinity of all T-cell epitopes and determined the thresholds at which approximately 80% and 50% of tested T-cell epitopes were predicted as binders. The number of false positives (FPs) at the thresholds was calculated for the four antigens and representative results are shown in Table To identify 80% of T cell epitopes, the threshold for each predictor had to be set low, which resulted in a large number of false positives. This problem was pronounced for predictors such as MHCPRED, MULTI_ANN and RANKPEP, since the number of false positives even exceeded that of true negatives. At this threshold, IEDB (consensus), MULTPRED (SVM), and PROPRED showed the best performance. On the other hand, the thresholds for predicting ~50% of known T cell epitopes were much more stringent, significantly lowering the rate of false positives relative to the 80% threshold. At this threshold, NetMHCIIpan, IEDB (consensus), and PROPRED showed the best performanceIn this study we evaluated the performance of 21 prediction servers for HLA-II binding peptides. Seven DRB1*0101 predictors, six DRB1*1101 predictors, three DRB1*0401 predictors, and one DRB1*0701 predictor showed good performance in identification of binders and non-binders.  None of predictors for DRB1*0301, DRB1*1301, and DRB1*1501 performed well, indicating that much room for improvement still exists for MHC-II prediction.The results suggest that some of current predictors are useful for pre-screening Th epitopes, although a relatively large number of false positives (at lower thresholds) and false negatives (at higher thresholds) would also be produced. Predictions using lower thresholds are useful for screening true negatives, while predictions at higher thresholds help cheaply identify a subset of the T-cell epitopes. Unlike MHC-I predictions, we have no evidence that nonlinear methods would perform better than linear methods. One possible reason may be due to the fact that nonlinear methods, such as ANN or SVM, generally require relatively larger amount of data for model development than linear methods. However, the amount of high-quality binding data for MHC-II binding is still far from sufficient, which limits the capability of nonlinear methods to recognize characteristics underlying MHC-peptide interactionIn summary, the prediction accuracy of HLA-II binding peptides is inferior to that of HLA-I binding peptides. Several factors appear to account for this disparity. Insufficient or low-quality training data has been the problem for developers of prediction methods for HLA-II binding peptides. Another problem with HLA-II predictions is the difficulty in identifying 9-mer binding cores within longer peptides used for training as well as lack of consideration of the influence of flanking residues. Amino acids flanking the binding core, contribute to MHC-peptide interactions and also antigen processing preferences ,40. AnotResults of this study will help researchers to determine the most appropriate servers for pre-screening of HLA class II binding peptides.  In addition, this study has defined basic criteria for slection of predication thresholds for selection of peptides that are most likely to be potential HLA-II epitopes. On the other hand, it provides guidelines for testing and test data to server developers. This knowledge, together with standardized test data sets should empower them to produce better solutions and improve prediction performance. Normalization and standardization methods that we introduced in this study enable annotation and integration of heterogeneous data into a uniform format, which facilitates the development of advanced algorithms. Future advancement in high-throughput measurements of binding affinities is expected to significantly improve the prediction performance of MHC-II binding peptides.vs. non-binders); d) Assess the prediction accuracy of promiscuous binding affinities; e) Assess the performance for predicting T cell epitopes.We evaluated 21 servers for prediction of HLA class II binding peptides that have been developed by 12 groups   and dog Each protein sequence was submitted to the prediction servers and the results were recorded. Most servers predict binding affinities of 9-mer peptides while the experiments were conducted on longer peptides ranging from 15 aa to 19 aa. Three mapping methods were explored to map the 9-mer predictions to experimental results. First, the highest prediction score of the overlapping 9-mer peptides spanning the length of a longer peptide was used as the predicted binding of the longer peptide. Second, the average score of the overlapping 9-mers was used as the predicted binding. Finally, the average of the top three predicted 9-mer scores of the overlapping peptides was used as the prediction score.ROC) [vs. false positive rate FP/(TN+FP) on the horizontal axis for the full range of the decision thresholds. The values AROC ≥ 0.9 indicate excellent, 0.9 > AROC ≥ 0.8 good, 0.8 > AROC ≥ 0.7 marginal and 0.7 > AROC poor predictions [Prediction accuracy is measured in terms of the area under the receiver operating characteristic curve (AROC) . The ROCdictions .50) lower than 100 nM , or lower than 1000 nM .In this study we defined promiscuous peptides as those peptides from the test set that bound four or more of the seven studied alleles. Binding was defined as half maximal inhibitory concentration (ICTo enable visual inspection for comparisons of predictions, both experimental measurements and predictions have been scaled to a common scale from 0 to 100 by linear transformation of the value ranges using the formula for each individual peptide:ymin is the minimum and ymax is the maximum score. The experimental binding affinity was corrected for variation in binding affinity of control peptides between different experiments then scaled. All values are accessible at DFRMLI site.where The authors declare that they have no competing interests. Previously HHL co-developed MHC_BPS, GLZ and VB co-developed MULTIPRED, and ELR co-developed Rankpep.VB and ELR designed the study, HHL performed the analysis, GLZ and ST collected and prepared data. HHL and VB drafted the article and all authors participated in manuscript.


The purpose of this study was to compare plasma levels of antioxidants and oxidative stress biomarkers in head and neck squamous cell carcinoma (HNSCC) patients with healthy controls. Furthermore, the effect of radiotherapy on these biomarkers and their association with survival in HNSCC patients were investigated.2α and ratio of oxidized/total ascorbic acid) were measured in plasma samples. Analysis of Covariance was used to compare biomarkers between patients and healthy controls. Kaplan-Meier plots and Cox' proportional hazards models were used to study survival among patients.Seventy-eight HNSCC patients and 100 healthy controls were included in this study. Follow-up samples at the end of radiotherapy were obtained in 60 patients. Fifteen antioxidant biomarkers  and four oxidative stress biomarkers , ferric reducing antioxidant power (FRAP) and modified FRAP were lower in HNSCC patients compared to controls and dietary antioxidants decreased during radiotherapy. Total hydroperoxides (d-ROMs), a marker for oxidative stress, were higher in HNSCC patients compared to controls and increased during radiotherapy. Among the biomarkers analyzed, high levels of plasma carotenoids before radiotherapy are associated with a prolonged progression-free survival . Additionally, high relative increase in plasma levels of d-ROMs  and high relative decrease in FRAP  during radiotherapy are also positively associated with survival.Biomarkers of antioxidants and oxidative stress are unfavourable in HNSCC patients compared to healthy controls, and radiotherapy affects many of these biomarkers. Increasing levels of antioxidant biomarkers before radiotherapy and increasing oxidative stress during radiotherapy may improve survival indicating that different factors/mechanisms may be important for survival before and during radiotherapy in HNSCC patients. Thus, the therapeutic potential of optimizing antioxidant status and oxidative stress should be explored further in these patients. Mammalian cells are constantly exposed to oxidative stress and rely on a comprehensive array of antioxidant defence comprising of both enzymatic and non-enzymatic molecules . Among nIt is hypothesized that oxidative stress is involved in the pathogenesis of different types of cancer -8. Head Radiotherapy is a cornerstone in the treatment of HNSCC. Ionic irradiation exposes all cells in the involved field to high levels of oxidative stress resulting in formation of ROS, increasing DNA damage and ultimately leading to cell death . AnotherThe nutritional status of HNSCC patients often reflects pre-diagnostic life style factors like smoking, drinking and low intake of fruits and vegetables, as well as changes in eating habits due to the tumour and adverse effects of radiotherapy. Although there have been advances in treatment, five-year survival rates in these patients has remained around 50-60% . Some reWe suggest that improved knowledge on the antioxidant and oxidative stress status, the impact of radiotherapy on this status, and its association with outcome could be useful for improving survival in these patients. Thus, in this study we have assessed an extensive profile of plasma low molecular weight antioxidants (endogenous and dietary) and oxidative stress markers before radiotherapy and monitored their respective response to radiotherapy in HNSCC patients. Furthermore, we have investigated how these parameters are related with survival.Eighty-seven patients with histologically verified HNSCC were recruited from Division of Cancer Medicine and Radiotherapy, Norwegian Radium Hospital, Rikshospitalet University Hospital in the period from May 2003 to May 2006. Seventy-eight patients were found eligible (two patients did not receive complete radiotherapy and 7 patients had previous history of cancer) in this prospective study. Follow-up samples at the end of radiotherapy were obtained from 60 patients. Of these 60 patients, twenty-four received assisted nutrition during radiotherapy.2 weekly and 6 fractions per week. One patient with nasopharyngeal carcinoma did not receive concomitant chemotherapy because of co-morbidity and high age. At the time of enrolment, concomitant chemotherapy was not the standard care at The Norwegian Radium Hospital.The patients received either post-operative radiotherapy or radiotherapy alone for a period of 5-7 weeks. All patients received external photon beam radiotherapy. The standard radiation technique used at the time of enrolment was computer tomography based conformal radiotherapy. Of the 78 patients included in the study, 50 patients received 70 Gy, 6 patients received 66 Gy, 1 patient received 64 Gy, 16 patients received 60 Gy (one died after 60 Gy) , 4 patients received 50 Gy and one patient died after 44 Gy. The patients receiving 70 Gy postoperatively had performed debulking surgery of the primary tumour and received 70 Gy to macroscopic lymph nodes in the neck and/or the primary tumour area. One patient received a split course accelerated hyperfractionated regimen with 1.5 Gy × 2 per day in 20 days (5 days per week). 76 patients received 2 Gy fractions 5 days weekly, but two of three patients with nasopharyngeal carcinomas received concomitant chemotherapy according to the Dahanca 14 protocol. The regimen used was cisplatin 40 mg/mFasting plasma from 100 control individuals, matched with respect to age range, gender and smoking status with HNSCC patients were selected. These controls were selected from a cohort of 356 participants in a validation study conducted at the University of Oslo . Participants were recruited after response to an invitation letter that was sent to a random selection of citizens in the Norwegian capital city and surrounding area. The aims of the validation study were first to validate a new food frequency questionnaire, and secondly to investigate normal ranges of nutrition-related biomarkers measured in blood samples. The distribution of participants intended to reflect the general population and few exclusion criteria were defined. However, participants with self-reported diseases were excluded before the control group for this study was selected.The basic characteristics of controls and patients are summarised in Table The study was approved by The Regional Committee for Medical Research Ethics. All the patients and controls gave their written informed consent.2α (8-iso PGF2α) and total hydroperoxides (d-ROMs), plasma was portioned in 1 ml and snap frozen in liquid nitrogen and kept at -80°C until analysis. Blood samples from controls were taken in EDTA (for carotenoids and tocopherols) and sodium heparin tubes .Blood samples taken before initiation of radiotherapy and in the last week of radiotherapy were collected in Stabilyte tubes and Vacutainer cell preparation tubes (CPT) containing sodium heparin as anticoagulant. Stabilyte tubes were centrifuged at 2800 g, 4°C for 10 minutes. The plasma obtained was prepared for reduced, oxidized and total GSH analysis as described in Sakhi et al. . The CPTThe plasma levels of reduced and oxidized GSH were analysed as described by Sakhi et al. . Total G2α was analyzed by HPLC using MS-MS detector [Total hydroperoxides (d-ROMs) were measured in plasma by a d-ROM kit as described by the manufacture . Plasma γ-glutamyl transpeptidase (GGT) was analysed using Vitros GGT slides with a Vitros 950 Chemistry system at the Department of Clinical Chemistry, Norwegian Radium Hospital, Oslo University Hospital. 8-iso-PGFdetector . One ml The redox potential of GSH in plasma was calculated using Nernst equation as described in Buettner et al. .The statistical program package SPSS  was employed. The biomarkers in pre-radiotherapy patients and controls were compared by independent samples t-tests. The groups were further compared, adjusted for age and body mass index (BMI) quartiles by Analysis of Covariance (ANCOVA). Paired t- tests were used to compare mean values before and after radiation therapy. Mean values are presented, and statistical significance was considered if p-value < 0.05.We have used Cox' proportional hazards model to compare the survival distributions of groups with high and low levels of plasma biomarkers , and to obtain estimated hazard rate ratios.To obtain adjusted hazard rate ratios, we tested the effect of a number of possible confounding factors: age, gender, smoking, treatment, stage, tumour localization and BMI. The confounding factors that changed the hazard rate ratios most were included in the final model. The number of confounding factors that could be included in the model depends on the sample size and we evaluated to have maximum 3 confounding factors in our study. Survival is shown by Kaplan-Meier plots, comparing patients with levels of biomarkers above and below the median.For the correlation analyses, Pearson and Spearman's rho correlation coefficient were calculated.For clinical outcome, progression-free and overall survivals have been used. Progression-free survival was calculated from start of radiotherapy until the date of first distant relapse, local recurrence, regrowth, regional lymph node metastasis, death from any cause or the last follow-up examination. Overall survival was calculated from the start of radiotherapy until death of any cause or last follow-up examination. Loco-regional control was calculated from start of radiotherapy until the date of local recurrence, regrowth, regional lymph node metastasis or the last follow-up examination.All tests were done two-tailed and p-values < 0.05 considered statistically significant.2α and ratio oxidized/total ascorbic acid (DHAA/TAA) were 73, 76, 76, 77, 77, 67, 76, 77 and 73 respectively. The number of samples for assessing the changes during radiotherapy for carotenoids, tocopherols, ascorbic acid, FRAP, modified FRAP, total GSH, total cysteine, GSH redox potential, d-ROMs, GGT, 8-iso PGF2α and ratio DHAA/TAA were 59, 59, 51, 57, 57, 57, 57, 50, 57, 59, 57 and 51, respectively. The numbers of control samples for analysis of tocopherols, FRAP, d-ROMs, total GSH and total cysteine were 97, 92, 99, 99 and 99, respectively. The remaining biomarkers had data for all patient and control samples.Due to different practical limitations, appropriate sample collection was not possible for every analysis in all patients and controls. Thus, the number of samples for pre-radiotherapy analysis for ascorbic acid, FRAP, modified FRAP, total GSH, total cysteine, GSH redox potential, d-ROMs, 8-iso PGFMean plasma levels of antioxidants and oxidative stress biomarkers in healthy controls and HNSCC patients (both pre- and post- radiotherapy) are shown in Table 2α and GGT increased 14%, 10% and 53%, respectively during radiotherapy , tocopherols and ascorbic acid, Table . The meaWith a median follow-up time for survivors of 28 months range 11-48 months), twenty-five patients are dead. Twenty-three patients died of HNSCC, one patient died of lung cancer and one patient died of unknown cause. Thirty-five patients have experienced progression: ten relapsed patients are alive, eighteen relapsed patients are dead, and seven patients are dead without previous relapse or regrowth of tumour. The projected three-year progression-free and overall survivals were 55% and 68%, respectively. Table 8 months,Above median values of several plasma carotenoids were significantly associated with improved progression-free survival. In univariate regression analysis of progression-free survival, high pre-radiotherapy plasma levels (above median) of total carotenoids and the single carotenoids  were significantly associated with prolonged progression-free survival Table . Plasma No association with survival was observed for any other antioxidant- or oxidative stress biomarkers.Patients with a high relative decrease in the plasma levels of total antioxidant biomarker FRAP during radiotherapy have a prolonged progression-free survival as shown in figure 2α also showed a trend with prolonged progression-free survival . After adjusting for confounding factors, the results were similar showing that there is a trend for 50% less risk of progression for patients with above median increase in plasma levels of 8-iso PGF2α during radiotherapy (Table Patients with a high relative increase (above median) in the plasma levels of oxidative stress biomarker d-ROMs have a prolonged progression-free survival Figure . The asspy Table .In order to investigate a combined effect of high levels of total carotenoids and high increase in d-ROMs compared to low levels of both these factors (above median levels compared to below median levels), we utilized a two-factor Cox model. The interaction term turned out non-significant and was removed from the model. The risk of progression  and overall death , respectively, were 80% and 87% less for the patients with high level of total carotenoids and high increase in d-ROMs as compared to low levels of both these factors.HNSCC patients have significantly lower levels of pre-radiotherapy plasma dietary antioxidants, FRAP and modified FRAP as compared to healthy controls; and dietary antioxidants further declined significantly during radiotherapy. The oxidative stress biomarker, d-ROMs, was significantly higher in patients as compared to controls and increased significantly during radiotherapy. Furthermore, pre-radiotherapy plasma carotenoids, relative increase in plasma levels of d-ROMs and relative decrease in plasma levels of FRAP during radiotherapy were positively associated with survival.While low levels of pre-radiotherapy plasma β-carotene, lycopene and vitamin E have been suggested in some preliminary studies involving HNSCC patients -17, the We also observed that these patients have high oxidative stress prior to radiotherapy as reflected by significantly higher plasma levels of d-ROMs as compared to healthy controls. To our knowledge, this is the first observation suggesting increased oxidative stress in newly diagnosed HNSCC patients before treatment.2α and GGT during radiotherapy.All the dietary antioxidants measured decreased after radiotherapy. The decrease in plasma dietary antioxidants during radiotherapy is possibly a combination of reduced intake of antioxidant-rich foods  and increased utilization of antioxidants by free radicals produced from ionising radiations. Bäckström et al.  has showWe have investigated the role of a number of plasma antioxidants and oxidative stress biomarkers on survival in HNSCC patients. Our results demonstrated that among different dietary antioxidant biomarkers, pre-radiotherapy plasma lutein, β-cryptoxanthin, β-carotene and total carotenoids are strong prognostic factors for progression-free and overall survivals. The risk of progression was 60% less in patients having plasma carotenoids above median levels as compared to patients with under median levels. After correcting for various confounding factors , the relative risk remained close to 50%. Meyer et el.  also shoWe have previously shown that high levels of post-radiotherapy plasma carotenoids  were positively associated with survival in HNSCC patients . Thus, p2α shows similar trend with survival after adjusting for confounding factors (p = 0.11). The risk of progression was decreased by 50% for patients with an above median increase in plasma levels of 8-iso PGF2α. Both d-ROMs and 8-iso PGF2α are biomarkers for oxidative stress, but measure total and specific oxidative damage in the cells, respectively. d-ROMs measure systemic oxidative damage caused by ROS to a number of organic substrates in a cell , but 8-iso PGF 2α measures a more specific oxidative damage mainly to polyunsaturated fatty acids in the cells. The therapeutic effect of radiotherapy mainly comes from production of ROS in tumour cells. Our results, thus, indicate that patients with an increase in systemic oxidative stress during radiotherapy had better progression-free survival suggesting that increased oxidative stress may be beneficial during treatment period. We also observed that the risk of progression and overall death, respectively, were 80% and 87% less for the patients with high level of total carotenoids and high increase in d-ROMs as compared to low levels of both these factors (above median levels compared to below median levels). The strong combined adverse effect of low total carotenoid level and low increase in d-ROMs may identify a high-risk group of patients that could be candidates for an intervention trial.Relative changes in plasma levels of d-ROMs and FRAP during radiotherapy showed an association with survival. The risk of progression developing distant relapse, local recurrence, regrowth or death was decreased by 70% and 60% for patients with an above median increase in d-ROMs and above median decrease in FRAP respectively, during treatment period. Relative changes in plasma levels of oxidative stress biomarker for lipid peroxidation, 8-iso PGFOur data shows that high pre-radiotherapy plasma total carotenoid status is beneficial for HNSCC patients. Plasma total carotenoids are biomarkers of fruits and vegetable intake. Plasma carotenoids may also reflect intake of carotenoid supplement. However, β-carotene supplements which increase plasma β-carotene significantly, show no beneficial effects on mortality and development of second primary tumours in head and neck cancer patients ,33, and Administration of carotenoid-rich fruits and vegetables may be more problematic during radiotherapy since oxidative stress mediates the therapeutic effects of radiotherapy and we observed that increased oxidative stress was beneficial during radiotherapy. To our knowledge, no study to date has studied the effect of increased carotenoid-rich fruits and vegetables during radiotherapy on the survival in this group of patients. Data from intervention studies with supplements (both α-tocopherol and β-carotene) in head and neck cancer patients during and after radiotherapy showed the supplement group had less severe side-effects of radiotherapy, but showed trends for increased rates of recurrence  that werOur data shows that plasma levels of pre-radiotherapy carotenoids in HNSCC patients are lower than healthy controls, decrease during radiotherapy and are positively associated with progression-free and overall survivals. High oxidative stress during treatment period is also beneficial for survival. The possible association between intake of carotenoid-rich fruits and vegetables and survival of HNSCC patients before, during and after radiotherapy should be investigated in future studies.2α: 8-isoprostagladin F2α; GGT: gamma-glutamyl transpeptidase; BMI: body mass index; ANCOVA: Analysis of Covariance; DHAA/TAA: oxidized/total ascorbic acid.HNSCC: Head and neck squamous cell carcinoma; FRAP: ferric reducing antioxidant power; d-ROMs: total hydroperoxides; ROS: reactive oxygen species; RNS: reactive nitrogen species; GSTs: glutathione S-transferases; HPV: human papillomavirus; GSH: glutathione; CPT: cell preparation tubes; 8-iso PGFRB has an interest in the companies Cgene AS, Bioindex AS and Vitas AS.Cgene and Bioindex were established by Birkeland Innovation, the Technology transfer office at the University of Oslo while Vitas was established by the Oslo Innovation Center. NEB works part time in the company Vitas AS. AKS worked part time for 2 months (January and February 2008) in the company Vitas AS.The other authors declare that they have no competing interests.AKS and KMR were responsible for sample collection, acquisition of patient data, statistical analysis, drafting and revising of the manuscript. AKS also executed the biomarker analysis in patient samples. MT was responsible for the statistical analysis and revising of the manuscript. NEB did the isoprostane analysis in patient samples. AK was responsible for the acquisition and analysis of control samples and revising of the manuscript. SS and RB planned the study and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/458/prepub


Because data from countries in Africa are limited, we measured the proportion of extensively drug-resistant (XDR) tuberculosis (TB) cases among TB patients in Burkina Faso for whom retreatment was failing. Of 34 patients with multidrug-resistant TB, 2 had an XDR TB strain. Second-line TB drugs should be strictly controlled to prevent further XDR TB increase. Extensively drug-resistant (XDR) tuberculosis (TB) represents an emerging public health problem worldwide, characterized by alarming fatality rates regardless of patients’ HIV status  on sputum samples from patients listed in the Burkina Faso Chronic TB Register. To be included in this register, patients must have experienced treatment failure after standard and retreatment regimens consisting of the following (in order): 2 months of streptomycin, rifampin, isoniazid, pyrazinamide, and ethambutol; then 1 month of rifampin, isoniazid, pyrazinamide, and ethambutol; and then 5 months of rifampin, isoniazid, and ethambutol. Failure was defined as a persistently positive sputum smear after 5 months of treatment. All patients were informed and consented to the study.Mycobacterium tuberculosis isolates by classic dilution method.From January 2006 through March 2009, a total of 156 patients with chronic TB were registered in Burkina Faso. For 88 patients, sputum samples were collected before treatment with second-line drugs; for 48, they were collected at 1–12 months of treatment with second-line drugs. Samples were immediately frozen and stored at –20°C before being transferred on dry ice for culture and first-line DST at the University of Brescia and for second-line DST and genotyping at San Raffaele Scientific Institute. Samples were cultured on an MGIT 960 automated system  according to the manufacturer’s instructions. DST to first- and second-line drugs was performed on all M. tuberculosis was isolated from 50 samples; 45 patients had not yet taken second-line drugs and 5 had. Of the 50 isolates, 34 (68%) were confirmed as MDR TB, 29 from untreated patients and 5 from treated patients. We identified 2 cases of XDR TB : 1 patient was sampled at month 24 of treatment with second-line drugs, and 1 was initially classified as having MDR TB before his disease progressed to XDR TB during follow-up treatment with second-line drugs.Patient 1 was a 33-year-old man who was born in Côte d’Ivoire, moved to Burkina Faso in 2000, and received a diagnosis of smear-positive pulmonary TB in July 2003. He received an 8-month standard treatment regimen (including 6 months of continuation with isoniazid and ethambutol). Treatment was directly observed during the first 2 months. In January 2004, his treatment was classified as failed, and he immediately started a standard retreatment regimen. Because his sputum did not clear by month 5, the patient was registered as a patient with chronic TB. During 2004, he traveled throughout Mali and Côte d’Ivoire. Back in Burkina Faso in January 2005, he was admitted to the reference national hospital for patients with chronic TB. His HIV test result was negative. At this time neither culture and DST nor a standard second-line drug regimen approved by the Green Light Committee were available in Burkina Faso. The patient was empirically prescribed kanamycin, ethionamide, ciprofloxacin, and pyrazinamide, which he took under direct observation as a hospital inpatient for 21 months; at discharge, his sputum samples remained positive.In December 2006, 2 sputum samples showed resistance to all first-line drugs; resistance to second-line drugs amikacin, ofloxacin, ethionamide, and cycloserine; and susceptibility to para-aminosalicylic acid and capreomycin. Appropriate and effective drugs were unavailable in the country. The patient was readmitted to the ward for patients with chronic TB and placed in a single isolation room. He died of TB in August 2008. Known contacts were monitored closely. A housemate was listed in the Chronic TB Register in June 2006. He traveled from Burkina Faso to Israel before DST could be performed. The patient’s sister-in-law died of MDR TB in March 2007; DST confirmed resistance to all first-line drugs but susceptibility to amikacin and ofloxacin.M. tuberculosis resistant to amikacin, kanamicin, ethionamide, and closerine but sensitive to ethambutol and ofloxacin. In September 2007, the patient started treatment with Green Light Committee–approved second-line drugs . Treatment was directly observed during the first 13 months. His sputum samples remained positive throughout treatment, and additional DST in October 2008 showed an XDR strain of M. tuberculosis. Mycobacterial interspersed repetitive unit genotyping showed an identical pattern for strains detected initially and during follow-up.Patient 2 was a 44-year-old man. His MDR TB was diagnosed in Côte d’Ivoire in June 2007, and he immediately moved back to Burkina Faso to seek care. His HIV test result was negative. Sputum samples collected in July 2007 grew MDR Our findings confirm that XDR TB can be found wherever the search capacity exists. Thus, despite unavailability of evidence for the widespread existence of drug resistance, high priority should be given to strengthening laboratory capacities in sub-Saharan Africa (Our study had 1 major limitation. Because our sample was a select population and included patients already receiving treatment with second-line drugs, we may have overestimated the proportion of XDR TB cases among MDR TB cases.Mechanisms of emergence of XDR TB in Burkina Faso differ from those in South Africa, where most identified XDR TB cases are primarily resistant, occur among HIV-infected patients, and result from exposure in the hospital or the community (Our study supports current policies for strictly controlled introduction of second-line drugs and the current Green Light Committee strategy that requires demonstration of compliance with guidelines for proper management of MDR TB before granting access to second-line drugs (Rapid adoption of a programmatic approach to management of MDR TB (


Under India's Revised National Tuberculosis Control Programme (RNTCP), >15% of previously-treated patients in the reported 2006 patient cohort defaulted from anti-tuberculosis treatment.To assess the timing, characteristics, and risk factors for default amongst re-treatment TB patients.For this case-control study, in 90 randomly-selected programme units treatment records were abstracted from all 2006 defaulters from the RNTCP re-treatment regimen (cases), with one consecutively-selected non-defaulter per case. Patients who interrupted anti-tuberculosis treatment for >2 months were classified as defaulters.1,141 defaulters and 1,189 non-defaulters were included. The median duration of treatment prior to default was 81 days (25%–75% interquartile range 44–117 days) and documented retrieval efforts after treatment interruption were inadequate. Defaulters were more likely to have been male , have previously defaulted anti-tuberculosis treatment .Amongst the large number of re-treatment patients in India, default occurs early and often. Improved pre-treatment counseling and community-based treatment provision may reduce default rates. Efforts to retrieve treatment interrupters prior to default require strengthening. India accounts for nearly 20% of the global incidence of TB, with an estimated annual incidence of 1.9 million tuberculosis (TB) cases The treatment outcome “default” under RNTCP is a patient who has not taken anti-TB drugs for 2 months or more consecutively after starting treatment In India, nearly one-quarter of TB patients are notified as “re-treatment”—i.e. with at least one-month history of previous anti-TB treatment We conducted a case-control study to understand the basic clinical and demographic characteristics of patients who defaulted from RNTCP re-treatment regimens, assess the patterns and timing of treatment interruptions, and evaluate risk factors for default.In India, patients are evaluated for TB using a standard diagnostic algorithm, starting with sputum-smear microscopy examinations for persons suspected of TB. Patients who are smear-positive are immediately initiated on treatment, and those who are smear-negative are evaluated further with antibiotic trial, repeat sputum examination, radiograph, and clinical evaluation.All patients diagnosed with TB are evaluated for history of previous anti-TB treatment and those with ≥1 month of treatment from any source are classified as re-treatment cases and prescribed the WHO-recommended Category II treatment, directly-observed and dosed thrice-weekly. RNTCP uses a 3-month (36 doses) intensive phase (IP) of isoniazid, pyrazinamide, rifampicin, and ethambutol (with streptomycin for the first 24 doses), and a 5-month (66 doses) continuation phase (CP) of isoniazid, rifampicin, and ethambutol. If a patient remains smear-positive after IP, then the same is extended by 1 month. Treatment duration is determined by the number of doses taken; missed doses lead to a longer total duration of treatment.A standard TB treatment card is maintained for every TB patient at the nearest health unit. The card records necessary contact, demographic, clinical, and treatment information, and completed by the treating medical officer.Adherence to RNTCP treatment is ensured for all TB patients by the use of directly-observed treatment (DOT), wherein an observer watches and supports the patient in taking their drugs. All patients are given a choice to choose their DOT provider who may be either a health worker in the public sector health-care facility or a community volunteer. Community volunteers may include local teachers, private providers, practitioners of traditional medicine, or anyone other than a family member. In the case of re-treatment patients, the need to provide injectable drug during the first 2 months of treatment limits the choice of DOT provider to those practitioners who are legally allowed to administer injectable drugs, i.e. public sector health-care workers, non-public sector health-care workers, or registered medical practitioners.The RNTCP has well defined guidelines on how to retrieve TB patients who interrupt treatment For this retrospective case-control study, cluster sampling was used to generate a nationally representative sample while retaining operational feasibility of data collection; clusters were the basic TB programme management unit (tuberculosis unit–“TU”) each covering ∼500,000 population. A sample size of 922 cases and 922 controls was determined to be sufficient to achieve 80% power (with a design effect of 2 used to account for single-level cluster sampling), with an odds-ratio >2 for the hypothesized risk factor of missing 5 doses prior to default, estimated to occur among 5% of non-defaulter controls. Given that on average 11 re-treatment patients defaulted per TU per year in 2006, we estimated that 90 TUs would be required to achieve the sample size. All 2,401 TUs were listed; 564 TUs (23.4%) were excluded due to security or operational reasons. From the remaining 1,837 TUs, 90 (4.9%) were randomly selected which were distributed in 73 districts across 15 of the 35 States/Union Territories.We defined ‘cases’ as patients registered in 2006 as re-treatment case (treatment category II) with a recorded treatment outcome of default from the TB register. RNTCP defines default as a patient who has not taken anti-TB drugs for more than 2 months consecutively any time after starting treatment The data sources used were TB treatment cards and TB registers. Retrospective data collection was limited to information available on routine records, including basic clinical and demographic information, DOT provider type, history of previous treatment, treatment adherence, adverse reactions, and retrieval actions. Treatment adherence was assessed from routine recording of missed doses on treatment cards. Data was abstracted from RNTCP TB registers and TB treatment cards by programme staff into Microsoft Excel, and then analyzed with SPSS 11.0  and Epi-Data 2.2. Differences in proportions between defaulters and non-defaulters were compared using the chi-square test (or Fisher's exact test if <10 observations were there in any cell). In multivariate logistic regression, we pre-selected variables for inclusion in the model based on prior information from studies from other settings on risk factors for default from new patients, with consideration for information available on routine records This retrospective analysis was conducted after review and approval by the Central TB Division, Directorate General of Health Services, Ministry of Health and Family Welfare, Government of India. The activity was determined to be programme evaluation of the implementation of national guidelines. Electronic databases created for this analysis were stripped of personal health identifiers and maintained securely.. Data could not be collected from 9 TUs due to operational reasons such as loss of treatment cards, TB registers or due to non-availability of staff for data collection. 160 (6.4%) patients were excluded from the analysis due to incomplete patient information (78 patients) or non-availability of treatment cards (82 patients). Of the 2,330 patients included in the final analysis, 1,141 patients were cases and 1,189 patients were controls .Out of 90 randomly selected TUs for this study, we received data from 81 (90%), encompassing 2,490 patients Of the 1,141 patients who defaulted from treatment, 907 (80%) were males table 1.); another 281 (25%) patients defaulted after completion of IP but before starting CP, and 140 (12%) patients defaulted during CP.The median duration of treatment prior to default was 81 days (inter-quartile range 44–117 days). Of 1,141 defaulters, 720 (63%) defaulted within 90 days of treatment, prior to completion of 36 doses in IP  of defaulters; an additional 87 (12%) patients had retrieval actions during the second week. Out of 726 patients who had any documented retrieval actions, 492 (68%) actions were taken by TB programme staff  were done by staff from the general health system. In only 11 (2%) instances was any retrieval action by medical officers (physicians) documented.Out of 1,141 defaulters, at least 1 retrieval action was documented in 726 (64%) patients. Relative to the last dose taken, the 1On bivariate analysis table 1,Treatment interruptions were more common among defaulters. Nearly 40% of patients who defaulted and >28% of the non-defaulters had at least 1 interruption of treatment during IP. Defaulters were more likely to have missed doses, than patients who did not default, regardless of the cut-off chosen for number of missed doses. Five or more missed doses (non-consecutive) or any 3 consecutive missed doses identified defaulters with 79% sensitivity, and 23% specificity.Multivariate logistic regression table 3 Although the practice was non-systematic, reason for default during the present retreatment regimen was recorded on the TB treatment cards of 735 patients (64.4%). Amongst these 735 patients, the most commonly cited reasons were migration 246 (21.6%), refusal 177 (15.5%), treatment from private sector 101 (8.9%), side effects 110 (9.6%), substance abuse 24 (2.1%), and others  77 (6.7%).More re-treatment TB patients are notified in India than any other country in the world, and default among this group is a serious public health problem Independent risk factors for default included male sex, registration as treatment after default, a history of previous anti-TB treatment outside of RNTCP, and DOT at a public health facility. The association of default and male sex has been previously reported for new patients, and it is unsurprising that previous default is associated with default during re-treatment no missed doses in IP documented prior to their default. Hence the usefulness of missing doses in any number or combination as a warning signal for potential default is limited.While missing doses during IP treatment was modestly associated with default, we found that in practice this would perform poorly as a signal for potential default. For example, nearly 40% of patients who defaulted missed at least one dose during IP, but so did 29% of non-defaulters. Viewed differently, 60% of patients who ultimately defaulted had The independent risk factors we identified were only weakly associated with default. These findings suggest that profiling registration data to identify and target re-treatment patients at risk for default is not likely to be widely successful. A similar observation on the limited usefulness of patient profiling was made from a case-control study in Hong Kong This is the first report of RNTCP retrieval efforts for patients who interrupted treatment and ultimately defaulted. Efforts to retrieve treatment interrupters were however found to be weak and far below programme guidelines. Zero retrieval actions were documented in 415 (36%) of the 1,141 defaulters. In those with documented retrieval action in more than half the patients it was delayed by more than 1 week. Contrary to RNTCP guidelines, staff of the general health system minimally participated in default retrieval efforts. For example, although programme guidelines are very clear that default retrieval is chiefly the responsibility of the treating medical officer, ∼2% of the documented retrieval actions were undertaken by medical officers. Instead, 68% of the documented retrieval actions were undertaken by the contractual TB programme staffs, who are few in number and cover large geographical areas. Actions and documentation of retrieval after interruption urgently require strengthening.By their nature, retrospective analysis based using routine records are subject to different types of information bias. The retrospective nature and the use of existing programme records limited our ability to assess the association of other risk factors which have been previously linked to default among new patients. For example, alcoholism and drug abuse have been previously associated with default in new patients a priori from our multivariate model.Treatment outcomes were not independently validated; if patients who had defaulted were misclassified as completed treatment in programme records, our ability to detect risk factors would have been diluted. The RNTCP, however, maintains a system of ongoing internal programme evaluation and review including periodic validations of recorded data and patient treatment outcomes. Thus widespread misclassification of treatment outcome is unlikely to have occurred. Our finding of a strong association between adverse events and default was likely influenced by reporting bias; hence, this finding should be interpreted with caution. The association between adverse events and default may be overestimated, and we were sufficiently concerned to exclude adverse events RNTCP has begun strengthening several areas to prevent and reduce default in re-treatment patients. RNTCP has enhanced it's monitoring of re-treatment outcomes in programme reviews and internal evaluations. To provide uniform pre-treatment patient education on the need for completing the full course of anti-TB treatment, emphasis during trainings on pre-treatment counseling has been coupled with the development of a standard patient information booklet, distributed in all languages with every box of RNTCP anti-TB drugs. To promote community-based treatment, the Central Unit has begun monitoring the proportion of patients treated by community-based DOT providers. Furthermore, local programme officers are being asked to provide greater emphasis on general health staff involvement with prompt retrieval actions by local providers and monitor treatment provision for patients who migrate during treatment.Defaulting from treatment is common among the large number of re-treatment patients in India. Default usually occurs early during treatment, particularly in the transition period from the IP to CP of treatment, and was weakly associated with male sex, prior default, prior treatment from a private provider, and DOT from a public health facility. Efforts to retrieve treatment interrupters fell far short of programme guidelines. Efforts to improve pre-treatment counseling, increase the proportion of patients treated by community-based treatment providers, and strengthen retrieval of treatment interrupters should yield immediate impact on default rates.This study, however, represents a starting point from where the RNTCP can begin to comprehensively seek to understand and reduce treatment default. Future prospective studies should seek to investigate the roles of other important risk factors that we could not assess in this retrospective analysis. Qualitative research to understand the challenges faced by patients and providers would further aid in pragmatic programmatic decision making.


We applied a subset of these sex-specific quality control filters to eight chromosomes in the Framingham Heart Study samples genotyped by Affymetrix 500 k SNP arrays, and identified another two single-nucleotide polymorphisms that failed to be picked up by the above global filters.We explored five sex-specific quality control filters in North American Rheumatoid Arthritis Consortium's Illumina 550 k datasets. Three X chromosome and three autosomal single-nucleotide polymorphisms flagged by sex quality control filters were missed by filters of call rate at 95% and Hardy-Weinberg equilibrium at 10 SNP filters such as call frequency, minor allele frequency (MAF), Hardy-Weinberg equilibrium (HWE), replicate concordance, and mendelian errors have been widely implemented to identify problematic SNPs. For example, the SNP thresholds used by Plenge et al.  for the Such SNPs, where data is differentially missing, miscalled, or both by sex may be of particular concern for diseases with differences in risk by sex, such as RA. In this analysis, we report on sex-specific differences in SNP call rates and genotype frequencies in the NARAC and Framingham Heart Study (FHS) data as released in Genetic Analysis Workshop 16 (GAW16).® Human Mapping 500 k Array Set. We used the 6,808 of FHS samples with a specified sex .Data were extracted from NARAC and FHS data files for GAW16 and analyzed using PLINK 1.04 . In the The overall sample and SNP statistics including observed proportion of heterozygotes (PropHet), call rate, and MAF were calculated using PLINK. We explored three approaches to identifying X chromosome SNPs that may be called differently by sex. The simplest were the proportion of male heterozygote calls and the absolute value of the difference in the call rates for males and females. The other, more statistical approach was to code all samples as female , use sex as the phenotype, and test whether the proportion of missing data was associated with sex (--test-missing option in PLINK ). We usep-values less than 10-7. This method flags SNPs with smaller differences between males and females when the overall call rate for the SNP is higher. Figure p-values less than 10-7.The call rates for males and females are shown in Figure p-values among controls greater than 10-6, there are 108, 90, and 3 of the SNPs that passed these tests had one, two, or three of the sex-difference flags. The three SNPs flagged by all three methods are shown in Table There are 151, 248, and 21 SNPs flagged exclusively by one, two, or three of these flags. After applying other QC filters of an overall call rate higher than 95% and Hardy-Weinberg -7. By utilizing the surrounding sequence for these three SNPs as provided by dbSNP, these three show significant alignment to both chromosomes 1 and Y, resulting in very similar BLASTn E-values and alignment scores [p-value less than 10-7 for the test of missingness by sex. There are 96 SNPs flagged by one or more of these thresholds . Table -6, there are three SNPs with two sex-difference flags and three with one flag.Figure t scores . Twenty-n = 51) with sex differences in call rate (>5%) and/or PropHet (>10%) on the chromosomes we examined . We flagged two such SNPs in the eight FHS autosomes we examined. Applied with care, sex-specific filters may be useful to identify and filter SNPs before the analysis of GWAS data.In the NARAC dataset, we flagged three SNPs on the X chromosome and three on autosomes with sex-specific calling errors that would not have been detected with general QC filters (95% call rate and 10EIRA: Epidemiologic Investigation of Rheumatoid Arthritis; FHS: Framingham Heart Study; GAW16: Genetic Analysis Workshop 16; GWAS: Genome-wide association study; HWE: Hardy-Weinberg equilibrium; MAF: Minor allele frequency; NARAC: North American Rheumatoid Arthritis Consortium; PropHet: Proportion of heterozygotes; RA: Rheumatoid arthritis; QC: Quality control; SNP: Single-nucleotide polymorphism.The authors declare that they have no competing interests.JEBW analyzed the FHS data. EWP proposed the study, drafted and critically revised the manuscript and suggested further analysis. HL analyzed the NARAC data and drafted the manuscript. KH reviewed results and suggested further analyses. All authors read and approved the final manuscript.


A record-linkage study was carried out between the Italian AIDS Registry and 24 Italian cancer registries to compare cancer excess among persons with HIV/AIDS (PWHA) before and after the introduction of highly active antiretroviral therapy (HAART) in 1996. Standardised incidence ratios (SIR) were computed in 21951 AIDS cases aged 16–69 years reported between 1986 and 2005. Of 101 669 person-years available, 45 026 were after 1996. SIR for Kaposi sarcoma (KS) and non-Hodgkin lymphoma greatly decreased in 1997–2004 compared with 1986–1996, but high SIRs for KS persisted in the increasingly large fraction of PWHA who had an interval of <1 year between first HIV-positive test and AIDS diagnosis. A significant excess of liver cancer (SIR=6.4) emerged in 1997–2004, whereas the SIRs for cancer of the cervix (41.5), anus (44.0), lung (4.1), brain (3.2), skin , Hodgkin lymphoma (20.7), myeloma (3.9), and non-AIDS-defining cancers (2.2) were similarly elevated in the two periods. The excess of some potentially preventable cancers in PWHA suggests that HAART use must be accompanied by cancer-prevention strategies, notably antismoking and cervical cancer screening programmes. Improvements in the timely identification of HIV-positive individuals are also a priority in Italy to avoid the adverse consequences of delayed HAART use. Three types of cancer that occur in HIV-positive individuals, namely Kaposi sarcoma (KS), non-Hodgkin lymphoma (NHL), and invasive cervical cancer (ICC), are currently included in the European clinical AIDS definition . HoweverAfter the introduction of the highly active antiretroviral therapy (HAART) in 1996, huge declines in KS and NHL incidence have been consistently reported in high-resource countries  were diagnosed with AIDS between 1986 and 2005; (2) were aged between 16 and 69 years at the time of AIDS diagnosis and (3) reported a ‘legal residence’ in areas covered by a CR. Person-years at risk were computed between 5 years prior to AIDS diagnosis, and the date of cancer or death or 10 years after AIDS diagnosis, whichever occurred earlier. This interval was left or right censored if no complete CR data were available for the corresponding years. To reduce losses to follow-up, dates of death were updated through record linkage with the National Mortality Database.Observed cases included incident cancer cases reported to CRs during the above-defined person-years at risk. Cancer site and type were classified according to the International Classification of Disease, 10th revision  and wereExpected numbers of different cancers were computed for each CR from sex-, age-, and period-specific incidence rates .SIR were calculated for calendar period, distinguishing the pre-HAART (1986–1996) from the post-HAART (1997–2004) period. For 1997–2004, and for cancers showing a significantly increased risk and at least 10 cases, SIRs were also computed separately by age group , HIV transmission category (injecting drug users (IDUs), men who have sex with men (MSM), heterosexuals) and time of cancer occurrence in respect to AIDS diagnosis . SIR were also computed separately by interval between first HIV-positive test and AIDS diagnosis , and country of birth .A total of 21 951 AIDS cases (78% men and 22% women) were reported in Italy between 1986 and 2005 in areas covered by a CR . The numA significantly elevated risk emerged in 1997–2004 for cancer of the liver  and penis , whereas the excess risk for leukaemia disappeared. Elevated SIRs for cancer of the anus , vulva and vagina , lung , brain , skin , HL , and multiple myeloma  in 1997–2004 were similar to those found in 1986–1996 . The comPersons with HIV/AIDS born outside Italy contributed 15% of person-years and 7.8% of cancer cases in 1997–2004. They showed similar SIR for AIDS-defining illnesses and slightly lower SIR of non-AIDS-defining cancers  than PWHA born in Italy (data not shown).Microscopic confirmation was available after 1996 for all ICC, anal cancer, and HL , as well as 79% of lung cancer. Eleven out of 16 liver cancers were microscopically or instrumentally confirmed. Microscopic confirmation was available for only one (a glioma) out of eight brain tumours, and seven had a concomitant AIDS-defining illness in the brain .For both AIDS- and non-AIDS-defining cancers the highest SIR emerged in the 3 months prior to or after AIDS diagnosis . Prior tSIR years for KS, NHL, and HL were lower among PWHA younger than 35 compared with older ones, whereas those for non-AIDS-defining cancers other than HL were higher . Women sPersons with HIV/AIDS who had less than 1-year interval between first HIV-positive test and AIDS diagnosis differed from other PWHA in many ways . Among tOur study showed substantial changes in the cancer pattern of Italian PWHA after the introduction of HAART in 1996. Non-Hodgkin lymphoma replaced KS as the most frequent cancer type and non-AIDS-defining cancers increased from 15 to 25% of all cancers. For the first time a significant excess of liver cancer emerged in the Italian AIDS linkage study  continued to be significantly increased among PWHA after 1996. The greatest cancer excess was found in proximity to AIDS diagnosis, but persisted in the 10 years afterwards. Notably, elevated SIRs were seen overall and in each HIV transmission category for cancers of the cervix and the anogenital tract that are, in the vast majority, associated with HPV infection . Hence, With respect to HL, our findings confirm previous reports , but an An increased risk for lung cancer among Italian PWHA was also confirmed (Skin cancer (non-melanoma) was increased by two-fold in PWHA as in previous reports  of information on the date of first HIV-positive test in the AIDS Registry allowed us, however, to focus on PWHA who had concomitant, or nearly concomitant, HIV infection and AIDS-defining illness. Such late presenters increased in Italy from 20.5% in 1996 to 55.5% of AIDS cases in 2007 . They weOur study showed that to prevent cancer in PWHA with increasing life expectancy, the use of HAART must be accompanied by more effective cancer-prevention strategies (