Mental disorders such as depression, anxiety and suicide represent an important public health problem in India. Elsewhere in the world a high prevalence of symptoms of common mental disorders have been found among people who inject drugs (PWID). Research in India has largely overlooked symptoms of common mental disorders among this high risk group. This paper reports on the results of a survey examining quality of life, depression, anxiety and suicidal ideation among adult males who inject drugs living in Delhi.Participants (n = 420) were recruited from needle and syringe programs using time location sampling and were interviewed using an interviewer-administered questionnaire. Self-report symptom scales were used to measure the severity of symptoms of depression (PHQ-9) and anxiety (GAD-2) within the preceding 2 weeks. We assessed the presence of suicidal thoughts and attempts within the past 12 months.The mean length of injecting career was 20.9 years indicating a sample of chronic injecting drug users, of whom only one-third (38%) were born in Delhi. The level of illiteracy was very high (62%), and just 2% had completed class 12. Scavenging / rag picking was the main form of income for 48%, and many were homeless (69%). One-third (33%) had been beaten up at least twice during the preceding 6 months, and many either never (45%) or rarely (27%) attended family events. We found a high prevalence of depressive  and anxiety  symptoms. Fifty-three percent thought about killing themselves in the past 12 months, and 36% had attempted to kill themselves.Our findings revealed a socially excluded population of PWID in Delhi who have minimal education and are often homeless, leaving them vulnerable to physical violence, poverty, poor health, imprisonment and disconnection from family. The high prevalence of psychological distress found in this study has implications for programmes seeking to engage, treat and rehabilitate PWID in India. Mental disorders are one of the leading contributors to the global burden of disease in both high and low income countries . Common Mental disorders constitute a major public health problem in India -9. EpidePrevious research has documented the co-occurrence of mental disorders and illicit substance use ,17. The A high prevalence of symptoms of depression, anxiety and suicidal ideation has been found among people who inject drugs (PWID) in high-income country settings using different diagnostic methods, instruments, cut-off values, and population samples -26. Few This paper reports on the results of a cross-sectional survey examining quality of life, depression and anxiety symptoms, and suicidal ideation among a community-based sample of adult males who inject drugs living in Delhi. The populations of PWID in Delhi, estimated to be at around 35,000  are fromThe research questions framing our study were: (1) what are the quality of life and socio-economic characteristics of this population of PWID?; (2) what is the prevalence of symptoms of depression and anxiety and suicidal ideation?; (3) what psychosocial factors are associated with symptoms of depression, anxiety and suicidal ideation?; and, (4) is there an association between risky injecting and sexual practices and symptoms of depression, anxiety and suicidal ideation? This paper will only report on the first and second research questions to allow for a full discussion of the characteristics of this population. The other objectives will be reported in depth elsewhere.In April and May of 2012, a cross-sectional survey was undertaken among PWID in Delhi using a structured questionnaire that was interviewer-administered. Ethics approval was provided in Australia by the Human Research Ethics Committee at The University of Melbourne (HREC 1137025). Local ethics approval was provided in India by the Institutional Review Board (IRB) at Sharan, The Society for Service to Urban Poverty. Sharan is an NGO that provides services to injecting drug users in New Delhi and is the partner NGO on this study.Eligible participants were those who were 18 years of age or older, had injected drugs at least once in the past month, were not currently enrolled in opioid substitution therapy (i.e. buprenorphine), and had given their informed oral consent to participate in the study. A consent form and plain language statement were orally communicated to potential participants, as literacy was limited for many participants. Consent was obtained orally after reading out the consent form. Participation in the study was voluntary and anonymous – no names or individually identifying information were recorded at any stage. Participants were provided with a small meal (e.g. chai and samosa) for their time in participating in the study. Participants were free to withdraw from the study during the questionnaire without losing access to the meal.Participants were sampled from three needle and syringe programmes (NSPs) in Delhi, each located in a distinctly different geographical area so that the sample was drawn from a diverse range of PWID from across Delhi; 1) Yamuna Bazar, 2) Nabi Karim, and 3) Jahangirpuri. Yamuna Bazar is located on the bank of the Yamuna River in central Delhi and is the base for a large proportion of the capital’s homeless and PWID. Jahangirpuri is a slum resettlement colony on the outskirts of metropolitan Delhi. The NSP at Nabi Karim is located in an urban ghetto in the Muslim quarter adjacent to New Delhi Railway Station.According to current data presented in the recent National AIDS Control Organisation (NACO) Annual Report 2011–2012 , it is eThe data were collected using Time Location Sampling (TLS), a recognized method for obtaining a probability-based sample from hidden populations -38. TLS Based on programme data, it was anticipated that there were twice as many PWID using the NSP in the Yamuna Bazar location in comparison to the other two locations. The selection of sampling events for the sampling calendar was weighted with probability proportionate to size, so that approximately half the sampling events would be drawn from the Yamuna Bazar location and the remaining half would be evenly split between Jehangipuri and Nabi Karim.The data collection team consisted of one field supervisor, one enumerator and four interviewers who were responsible for recruiting and interviewing participants. During sampling events, the enumerator would use a “counter” to count every male who used the NSP to obtain new needles/syringes. Interviewers were directed by the enumerator to approach potential participants immediately after they had obtained new needles / syringes from the NSP. Not all people using the NSP during a sampling event could be approached to participate given there was a limited number of interviewers. Additionally, there would be more people attending the NSPs during some sampling events than others, thus creating a scenario of unequal probability of selection for participants across sampling events. The enumerator counted all people actually interviewed during each sampling event in addition to counting those who had used the NSP to enable the construction of selection probability weights as discussed in the data analysis section below. Additionally, the enumerator collected data on the number of people who were approached yet declined to participate and the number of participants who terminated the interview early.The questionnaire was interviewer-administered to maximize the quality of the data collected. The questionnaire had five domains: 1) background information including socio-demographics, 2) drug use practices, 3) sexual behaviour, 4) quality of life, and 5) symptoms of depression, anxiety and suicidal ideation. It was anticipated that many of the participants would have never received any schooling. As a result, the questionnaire was kept as brief and simple as possible and it took approximately 30 minutes to complete. The questionnaire was translated into Hindi, back-translated into English and then piloted in the field to ensure equivalence and appropriateness of the questions and scales. Local research assistants were used for the data collection phase, all of whom had previously been involved in administering questionnaires with PWID. The research assistants were trained in using the questionnaire and ethical research conduct and were closely supervised by an experienced local research officer.Questions regarding demographics, drug use and sexual behaviours (predominantly condom use with various partners) were adapted from previous research  among inFour generic questions relating to quality of life were asked, covering four different aspects of quality of life. The questions were adapted from themes contained within the WHOQoL scale  and the Symptoms of depression were measured using the Patient Health Questionnaire (PHQ-9), a nine-item screening tool based on criteria for depressive disorders in the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) ,45. The The conventional cut-off is ≥10, yielding a sensitivity of 88% and a specificity of 88% for major depression . The PHQSymptoms of anxiety were measured using the Generalised Anxiety Disorder (GAD-2) scale, a two-item ultra-brief screening tool that follows the same format and response options as the PHQ-9 . Scores Suicidal ideation was captured using three questions adapted from the Suicide Behaviours Questionnaire (SBQ) . QuestioWith an assumed prevalence of psychological distress of 50%  and a 95% confidence interval with a 5.5% margin of error, it was calculated that a sample size of 312 PWID would be required. TLS sampling is similar to cluster sampling, as randomly selected ‘blocks of time and space’ are potentially clusters of homogeneity. The required sample size was increased to 420 after adjusting for a design effect of 1.25.Data were entered into SPSS version 20 and cross-checking was undertaken among 10% of questionnaires to ensure there were no systematic errors when entering the data. Preliminary tables were generated for each variable to check for invalid entries. Data were analysed using Stata version 10. Survey commands in Stata were used to account for the TLS sampling design, specifically the effect of clustering within sampling events and the probability that a person was sampled ,56. PartDescriptive analysis produced adjusted proportions and means with their respective 95% confidence intervals (C.I.). The range and median were used to provide important information as to the distribution of the continuous depressive and anxiety symptom measures. Because these statistics could not be generated using survey commands in Stata, due to the limited options available in these commands, they were generated without adjustment for the sampling design.There were 420 PWID recruited into the study: 220 (52%) from Yamuna Bazar, 100 (24%) from Nabi Karim and 100 (24%) from Jehangipuri. During recruitment there were 173 PWID who were approached but declined to participate; if we take a conservative assumption that all 173 met the inclusion criteria, this would indicate a response rate of 71% (420 ÷ (420 + 173)). There were no occasions where a participant terminated the interview before it was completed.Table Tobacco was consumed daily for the majority of these PWID (93%). Few (7%) consumed alcohol daily. The mean age of first injection was 15.8 (95% C.I. 15.3-16.3) and one-third (34%) had first injected at the age of 13 or younger. The mean length of injecting career was 20.9 years (95% C.I. 19.8-22.0). The most commonly used opioids in the last three months were buprenorphine (97%), heroin (77%), Spasmoproxyvon (27%) and pentazocine / morphine (25%). Buprenorphine, Spasmoproxyvon and pentazocine / morphine are synthetic opioid analgesics commonly found in chemist shops. Spasmoproxyvon is the brand name for a compound drug containing dextropropoxyphene, dicyclomine hydrochloride and paracetamol. Other commonly used drugs were marijuana (73%), diazepam (53%) and antihistamines . The main drug injected in the last three months was typically buprenorphine (77%) or heroin (18%), both of which were commonly injected in a cocktail with a liquid antihistamine and often diazepam as well. Few (14%) PWID were injecting only once a day or less.Table The level of illiteracy was very high (62%) and just 2% had completed 12 years of primary and secondary education. On average, PWID in this community live off a mean of 193.5 rupees per day (95% C.I. 181.1-206.0), roughly $US3.50 and approximately half had a daily income of ≤100 rupees (22%) or 101–150 rupees (27%). Scavenging / rag picking was the main form of income for 48% of PWID and just under one-third did either unskilled manual work (19%) or skilled manual work (11%). A small group of PWID primarily generated their income from either crime (6%) or begging (2%).There was a very high proportion of PWID who were homeless (69%) and several others mostly slept in insecure housing including rented rooms (6%) and other temporary housing (4%). Seventy percent had a history of imprisonment. Experiences of physical violence were common: 33% had been beaten up at least twice during the preceding six months and 11% had been beaten up four or more times.Table Among the nine items on the PHQ-9, the highest average score was for the item “feeling bad about yourself – or that you are a failure or have let yourself or your family down”; the mean was 2.2 (95% C.I. 2.1-2.3). The proportion of participants who received the maximum score of 3 (i.e. had this symptom nearly every day in the last two weeks) for this item was 51% (95% C.I. 44%-58%).The mean GAD-2 score was 3.4 out of a total possible score of 6, with a range of 0 to 6 and a median of 4. Seventy-one percent qualified as having anxiety symptomatology (cut-off score of ≥3). There was a small group  of PWID who displayed no recent (i.e. past two weeks) symptoms of either depression or anxiety.Suicidal ideation during the past 12 months was common. Fifty-three percent thought about killing themselves and almost one-quarter either often (15%) or very often (7%) thought about killing themselves. A substantial proportion reported to have actually attempted to kill themselves (36%) in the past 12 months.The findings from this study profile an impoverished, vulnerable and isolated population of men who inject drugs and whose lives are shaped by a significant level of psychosocial distress. The average age of participants was in the mid-30s with few aged below 25 and most had commenced their injecting careers during adolescence, indicative of a population of PWID who belong to the more chronic end of the addiction spectrum. The prevalence of symptoms of depression and anxiety was very high and suicidal thoughts and acts were disconcertingly common.More than half the participants were born outside of Delhi, suggesting many of these men were part of the phenomenon of large-scale migration into the city whether as adults or children. Migration into Delhi has fuelled a population growth rate of between 4-5% per year between 1951 and 2001 , making In our study, the quality of life and socio-economic indicators reveal a socially excluded population of PWID in Delhi who have minimal education and are often homeless, leaving them vulnerable to physical violence, poverty, poor health and imprisonment. These men are clearly excluded from the rapid social and economic progress around them.The social exclusion extends to family relationships, the core of the social fabric in India , with maThere was a high prevalence of symptoms of depression and anxiety among this community-based population of men who inject drugs. In our study, 84% (PHQ-9 ≥10) had symptoms of depression, 54% (PHQ-9 ≥15) had moderately severe or severe symptoms of depression and 71% (GAD-2 ≥3) had symptoms of anxiety. The most common symptom of depression was feelings of worthlessness and self-blame which is not very surprising given the highly stigmatised nature of injecting drug use, the disconnection with family and the deprived socio-economic circumstances for many among this population, but is in contrast to the findings of a population-based survey of depression in Chennai that also used the PHQ and reported depressed mood as the most common symptom .These results are somewhat comparable to studies conducted in high-income countries among PWIDs using self-report symptoms scales, despite variation in study settings and the particular scale used. Lemstra et al.  used theSimilarly high prevalence rates have been found in other studies conducted in the Asian region. Gu et al.  used theOur study revealed a disconcertingly high prevalence of suicidal ideation. We found that 53% had thought about killing themselves in the past 12 months and 34% had made a plan to kill themselves. This result is the same as that found by Sarin et al.  who measOur study also found that many (36%) men who inject drugs in Delhi had moved beyond thinking about killing themselves to actually attempting to kill themselves in the past 12 months. Suicide is a real option for these men, with at least one means readily available in the form of a drug overdose. Chen et al.  also fouThe high prevalence of psychological distress found in this study has implications for programmes seeking to engage, treat and rehabilitate PWID in India. Two broad approaches appear to be worth further consideration, although both face substantial challenges. One approach to integrating mental health into services for PWID would be to adopt a conventional psychiatric biomedical model based around the use of screening tools and referral mechanisms complimented by the development of mental health literacy ,62 and mHowever, there is a question mark over whether conventional screening and treatment approaches would be effective for people who live in such adverse circumstances. Separating human distress from a diagnosis of depression or anxiety is difficult . FurtherWhilst severe depression and anxiety may require psychiatric treatment, those with milder to moderate symptoms could be better served by the adoption of a broader public health approach that targets the population rather than the individual . The conThe use of NSPs as the primary site of participant recruitment may have created a sampling bias. Those PWID who participated in this study are more likely to be known to services and may represent the more chronic end of the addiction spectrum. The method of participant recruitment may have missed out on casual users and those who were newer to injecting drug use. Additionally, those who declined to participate may differ from those who did participate and we are unable to assess the degree to which our participants were representative of the broader NSP client group; however, a response rate of 71% is acceptable and suggests the sample is satisfactorily representative.Symptoms of depression and anxiety were measured using self-report screening tools rather than a formal psychiatric diagnosis. The selection of the optimal tool for evaluating comorbid psychiatric conditions in drug using populations remains controversial. In our study, it is not possible to differentiate between psychiatric symptoms that are independent of substance use and those that were a consequence of it. Furthermore, the high level of psychological distress among our study participants is not surprising given their significant socioeconomic stress, disconnection from family, and the stigma connected to illicit drug addiction. Jacobs  argues tOur study into men who inject drugs in Delhi revealed an impoverished, vulnerable and isolated population whose lives are shaped by a significant level of psychosocial distress. The prevalence of depressive and anxiety symptoms among this population of men who inject drugs in Delhi was very high and suicidal thoughts and acts were disconcertingly common. Given the above and the high level of unmet need for mental health care in India, further research and discussion is required to inform the most appropriate public health response to the mental health and wellbeing needs of this population group.The authors declare that they have no competing interests.GA and MK designed the study and conceptualized the analysis in this paper. GA developed the sampling strategy and supervised the implementation of data collection in collaboration with LS, SS and AM. AM entered and cleaned the data under the supervision of GA. GA undertook the data analysis and wrote the first draft of this paper. LS and SS provided links to the injecting drug user community and data collection sites, and contributed fundamentally important contextual knowledge that informed the development and implementation of the study. All authors read and contributed to the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/13/151/prepub


Background. Missing data are a significant problem in health-related quality of life (HRQOL) research. We evaluated two imputation approaches: missing data estimation (MDE) and assignment of mean score (AMS). Methods. HRQOL data were collected using the Medical Outcomes Trust SF-12. Missing data were estimated using both approaches, summary statistics were produced for both, and results were compared using intraclass correlations (ICC). Results. Missing data were imputed for 21 participants. Mean values were similar, with ICC >.99 within both the Physical Component Summary and the Mental Component Summary when comparing the two methodologies. When imputed data were added into the full study sample, mean scores were identical regardless of methodology. Conclusion. Results support the use of a practical and simple imputation strategy of replacing missing values with the mean of the sample in cross-sectional studies when less than half of the required items of the SF-12 components are missing. Health-related quality of life (HRQOL) is an increasingly important outcome in both clinical trials and observational studies –4. It isMissing data are a significant problem in HRQOL research due to the potential loss of statistical power as the sample size is reduced and, more importantly, due to the potential for bias , 6. For The potential impact of the problem has been well described , includiThe Medical Outcomes Trust SF-36 and the shorter SF-12 health surveys are the most widely used HRQOL assessments in the world, with translations into 138 and 113 languages, respectively . When scOne practical approach to this problem has been put forward by Perneger and Burnand . They haPerneger and Burnand were able to test the difference between the imputed and the true score; however, in reality, the reason for imputation is that the true score is not available. In a recent randomized clinical trial of two approaches to compression bandaging for leg ulcers , small aWe sought to compare the PCS and MCS scores generated using MDE  with thocross-sectional baseline administration of the SF-12 are reported here. The survey was either completed by the participant independently during their regularly scheduled nursing care visit or completed with the help of a caregiver. Ethics approval for the RCT was granted by the Queen's University Research Ethics Board (REB# NURS-140-03), and local approval was granted at individual sites where required.SF-12v2  was collStudy personnel were licensed to use the MDE  to estimComplete SF-12 data were available for 384 (90.6%) of the 424 participants. However, 23 (5.4%) were missing one or more items and 17 (4.0%) missed the entire questionnaire. These 17 were not included in the imputation by MDE or AMS. Two of the 23 were not included since one had too much additional information missing for MDE assignment and the other was randomized after MDE had been completed.For the remaining 21 respondents, 20 missed 1 item and 1 missed 2 items for a total of 22 missing items. Of these, most were in the physically oriented domains (16/22). The first general health item (gh01) was missed twice; each of the physical function items  was missed 5 times; the first role physical item (rp02) was missed 4 times. For the mentally oriented items, the second role emotional item (re03) was missed once, the first mental health item (mh03) was missed twice, and the vitality item (vt02) was missed 3 times. This suggests that individual items, rather than entire sections, were missed; the one person who missed two items missed a physical function and a mental health item.These data support the use of a practical and simple imputation strategy of replacing missing values with the mean of the population under study when less than half of the required key items of the SF-12 components are missing in cross-sectional research. This approach is not suitable for cases where the entire questionnaire is missing, where there is a clear pattern of missing data, or for data missing longitudinally, where more advanced statistical methodology should be utilized , 8–12. MApplication of the group mean for an individual item will result in some regression towards the mean, particularly for those with extreme scores , 6. ThisThis study is limited by the small sample size, by the fact that participants are from a single study, and most had only 1 item missing; it is possible that the comparison might have turned out differently had many been missing 2-3 items. However, the results are not intended to be definitive; rather, they are intended to provide additional support for the larger study which proposed this methodology .Obviously the best approach to missing data is to prevent it by making every attempt to obtain complete data. However, even the most carefully designed studies may still have missing data, even when specific interventions to minimize missing data are used , particu


Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis.Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases.Using a panel of serum from A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this zoonosis is suspected. Fasciola hepatica-infected patients, we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. The test is easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with 99.9% sensitivity and 99.9% specificity. This ELISA will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this zoonosis is suspected.Fascioliasis is a food-borne human disease caused by helminth parasites of the genus Fasciola. It is a global disease of domestic animals but its increased recognition as a major zoonosis has led to the World Health Organization including fascioliasis on the list of important human parasitic diseases. Current diagnosis of human fascioliasis involves the detection of eggs in the stool. However, eggs are not observed during the acute phase when the parasite is migrating through the tissues, and can be missed during the chronic phase when parasites are in the bile duct due to the sporadic release of the bile into the intestines. Using a panel of serum from  Fascioliasis, or liver fluke disease, is a food-borne infection caused by trematodes of the genus Fasciola. The disease has been traditionally viewed as of mainly veterinary importance because of the substantial production and economic losses it causes in livestock, particularly sheep and cattle. In contrast, human fascioliasis has until recently been neglected by the medical community. Due to its increased spread and chronic nature, it is now recognized as a disease of global human concern by the (WHO) Fasciola hepatica has a worldwide distribution and causes major health problems in Europe , the Americas , Cuba and Oceania and overlaps with F. gigantica in many areas of Africa and Asia Fasciola gigantica, in humans was thought to be of relatively little importance due to its low incidence in endemic areas. However, since fascioliasis is not a reportable disease in many countries, the number of cases (>500) reported in the literature represent the tip of the iceberg F. hepatica tolerates a wide range of environmental conditions and has a remarkable ability to adapt to new hosts F. hepatica is also related to the geographic expansion of its original intermediate host, the snail Galba truncatula. By contrast, the smaller geographic distribution of F. gigantica seems to be related to the weaker diffusion capacity of its intermediate snail hosts Fasciola spp. have been estimated to produce 20 000 to 50 000 eggs per fluke per day in animals Infections in animals and humans occur when vegetation or water contaminated with infective encysted dormant larvae (metacercariae) is ingested. The parasites excyst in the host intestine, migrate through the intestinal wall into the peritoneal cavity and then into the liver parenchyma where they caused extensive tissue damage and blood vessel hemorrhaging representing the acute phase of the disease F. hepatica infections are asymptomatic and disease may appear anywhere from a few days to several years after infection thereby making the diagnosis challenging Up to 50% of Fasciola antibodies provide an alternative to coprological examination. Anti-F. hepatica antibodies can be found after 2–4 weeks post-infection providing a means for early detection of disease using parasite extracts or excretory-secretory (FhES) products as antigen Enzyme-linked immunosorbent assay (ELISA) methods developed for determination of anti-26). We also employed more recently developed and commercially available secondary antibodies against anti-total IgG and several subclasses  to screen and optimize the test using a panel of serum samples well-characterized Fasciola-infected patients. The test we developed is easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with 99.9% sensitivity and 99.9% specificity. This ELISA will be a useful standardized method not only for testing individual samples but can be employed in mass screening programs to assess the extent of human fascioliasis in regions where this zoonosis is suspected.Previous studies used native FhCL1 or an enzymatically active recombinant rFhCL1. However these were prone to breakdown and auto-catalytic degradation during purification and also cleaved immunoglobulin in the ELISA. We have therefore employed an inactive recombinant FhCL1 variant , Peroxidase-conjugated labeled anti-human immunoglobulin IgG (Goat) was from Perkin Elmer . The substrate 3,3′,5,5′-Tetramethylbenzidine (TMB/E) was purchased from Millipore . Peroxidase-conjugated anti-human immunoglobulin IgG1, IgG2 and IgG4 were purchased from Southern Biotech .F. hepatica and clinically diagnosed in the hospital, samples from 72 Cuban and 63 Canadian individuals that were shown to be negative for Fasciola infection, and 158 serum samples obtained from individuals infected with other parasitic diseases including, amoebiasis (12), ascariasis (10), Chagas disease (10), cysticercosis (10), echinococosis (13), enterobiasis (2), filariasis (11), giardiasis (5), leishmaniasis (9), malaria (14), metorchiasis (9), schistosomiasis (9) , strongyloidiasis (6), toxocariasis (14), toxoplasmosis (13), and trichinellosis (11).The human Fasciola samples were reviewed and approved by the ‘Pedro Kourí’ Tropical Medicine Institute  Biomedical Research Ethics Board. The human control and other parasitic diseases sera were obtained from the Passive Parasitic Diseases Surveillance System diagnostic testing at the National Reference Laboratory for Parasitology  and were considered exempt. All samples used in this study were anonymized. These consisted of samples from 93 Cuban individuals that were coprologically-positive for eggs of F. hepatica preprocathepsin L1 cDNA was previously cloned in our laboratory into a P. pastoris multicopy system using P. pastoris GS115 strain and pPIC9K vector (26). The variant FheproCL1Gly26 (Cys26 to Gly26) was used in this study and expressed as described by Collins et al. (21). The inactive enzyme was produced by fermentation at 30°C and 250 rpm in 1 liter BMGY broth buffered to pH 6.0 into 4 liter baffled flasks until an OD600 of 2–6 was achieved. The cells were centrifuged at 3,000× g for 10 minutes at room temperature and the induction initiated by resuspending the pellets in 200 ml BMMY broth and adding 1% of filter–sterilized 100% methanol every 24 hours for 3 days. The culture was then centrifuged at 16,000× g for 30 minutes at room temperature. The pellets were discarded and FhCL1 isolated from the supernatant by Ni-NTA affinity chromatography as previously described The full length For the purpose of optimizing the ELISA a pool of serum from fasciola-infected individuals (30) and of negatives controls (30) was prepared. Determination of the optimum antigen concentration and the dilution of the sample serum and secondary conjugated antibody that gave the most superior background-to-signal ratio were assessed by employing a matrix formation. Using different 96-well plates, each with a constant antigen concentration, different dilutions of the pooled positive control serum was added to the wells from top to bottom (well A–G) while different dilutions of the secondary antibody were tested in duplicate from left to right (wells 1–12). All optimization experiments were repeated at least once.For each plate FhCL1 antigen was dissolved in bicarbonate/carbonate coating buffer at pH 9.0. One hundred microliters of the solution was then added to each well and incubated overnight at 4°C. After washing four times, excess protein binding sites were blocked at 37°C for 1 h by adding 100 µl of 2% bovine serum albumin diluted in PBS/0.1% Tween 20. After a further washing procedure, 100 µl of pooled samples sera  were added and the plate incubated for 1 h at 37°C. Following another wash, 100 µl of peroxidase-conjugated anti-human IgG  was added to the wells and the plates were incubated for 30 min at 37°C. After a final washing step bound antibodies were detected by the addition of 100 µl of TMB. The color was developed for 10 min and the reaction was stopped with 50 µl of 0.1 M sulphuric acid. The plates were read on an ELISA plate reader at 405 nm. All serum samples were analyzed for the binding of total IgG and IgG1, IgG2 or IgG4 using the appropriate secondary monoclonal antibodies specific for each. Box-Cox transformation of the data from uninfected control for IgG showed that lambda of 0.33 minimized skewness in the data. Data was therefore transformed by cube-rooting to normalize the distribution prior to statistical analysis F. hepatica and a pool of sera from 20 Cuban patients negative for this parasitic infection. We performed a matrix comparison of ELISAs using various antigen concentrations, dilutions of the pooled primary sera and dilutions of secondary antibodies specific for different human antibody isotypes. The ELISA conditions providing the best positive to negative signal ratio and used in our subsequent studies were as follows: wells were coated with 100 µl of 0.25 µg/ml of the FhCL1 antigen; the dilution of the human primary sera used was 1/200 and the dilution of the secondary antibody was 1/32000, 1/8000, 1/100 and 1/32000 for secondary antibodies anti-total IgG, anti-IgG1, anti-IgG2 and anti-IgG4, respectively.To determine the optimal conditions for diagnosis of human fascioliasis by ELISA using the FhCL1 as antigen we used a pool of positive control sera from 20 Cuban patients with a known infection with A total of 386 serum samples were screened using our optimized ELISA. Statistical analysis of the ELISA data was performed to evaluate the efficacy of FhCL1 to discriminate between positive infected individuals and negative non-infected individuals. First, the results for assays using anti-total IgG as the secondary antibody were plotted in a histogram to evaluate the distribution of the population to be analyzed . The datStatistical analysis of the data obtained using anti-IgG4 as the secondary antibody was also performed. The data was plotted into a histogram and results analyzed. Two of the negatives samples do not seem to belong to the distribution of the rest of the negatives and are outside the cut-off that discriminate 99.99% of the uninfected patients . To grouWhen we employed secondary antibodies specific for IgG1 and IgG2 in our ELISA assays the sensitivity and specificity dropped drastically compared to anti-total IgG and anti-IgG4. For these assays we found that the data was badly skewed from a normal distribution and a clear cut-off between the negative and positive patient sera could not be established. Thus a definitive distinction of non-infected and infected patients could not be made .F. hepatica negative and positive patients although the gap was small, as found with the histogram. Both anti-IgG1 and anti-IgG2 were even less effective with a large overlap between the Fasciola-negatives giving the highest readings and the Fasciola-positives giving the lowest readings  and non-infected (72 Cubans and 63 Canadians) and compared these with sera obtained from patients infected with range of other worm as described above. We used anti-total IgG and anti-IgG4 as the secondary antibodies .F. hepatica closely matched that obtained with the negative control samples. We found that using 0.55 OD units as cut-off with anti-total IgG as secondary antibody, the test can discriminate between F. hepatica patients and all other infections examined  was added F. hepatica (diagnosed by eggs in stool) and a pool of negative sera from matched non-infected patients. The optimization of the ELISA allowed us to determine the best dilution of the primary sera and secondary antibody to obtain an excellent discrimination between positives and negatives using secondary antibodies that detected total IgG and IgG4. Either of these reagents could be used to diagnose individuals, as proven in previous studies Optimization of the ELISA was performed by using a pool of sera from patients infected with When using anti-IgG4 as the secondary antibody, two patients  appeared as borderline cases; these did not group to the normal seronegative distribution and were beyond the cut-off. In an attempt to include them in the seronegative group the cut off was set at 0.1 OD units and consequently we obtained a narrower gap between positive and negative groups compared to anti-total IgG.et al.Previous studies by O'Neill We also analyzed the data derived from ELISAs that employed secondary antibodies specific to IgG1 and IgG2 isotypes. However, these secondary reagents did not perform satisfactorily and several patients were misdiagnosed. An increase in the background was observed when using anti-IgG1 and not all Fasciola-infected patients produce IgG1 antibodies. This resulted in an overlapping of some Fasciola-negative and Fasciola-positive sera decreasing the sensitivity and specificity of the test considerably. This was not surprising due to the fact that Fasciola infection induces the production of IgG4 followed by IgG1 and to a lesser extent IgG2 and IgG3 F. hepatica eggs can be confused with eggs from other helminths. Therefore, a good diagnostic test needs to be able to distinguish between Fasciola and other parasitic diseases. We screened human samples infected with different parasitic diseases and analyzed the ELISA data statistically. Using our ELISAs no cross-reactivity with other parasitic diseases was observed; in fact, the mean absorbances observed for the various diseases examined were not significantly different from the non-infected negative controls patients regardless of whether we employed anti-total IgG or anti-IgG4 secondary antibodies. Moreover, all the Fasciola-infected individuals had significantly higher absorbance readings than those obtained from patients infected with the other parasites. This is consistent with previous studies using native cathepsin L1 Sera sample from patients infected with other diseases were used to evaluate cross-reactivity in our ELISA. Analysis of cross-reactivity is extremely important since fascioliasis is a worldwide parasitic disease which can co-exist with other human parasitic diseases which can complicate diagnosis. Furthermore, current parasitological methods depend on the expertise of the worker because Over the last two decades there has been a renewed interest in human fascioliasis. This is due to the increase in epidemiological surveys that has revealed the present emergence/re-emergence of the disease both in humans and animals in many regions P. pastoris culture medium allowing us to provide sufficient quantities of material to supply diagnostic centers for mass screening in regions where human fascioliasis is prevalent.Here, we have produced a standardized test using a highly stable recombinant form of cathepsin L1, FhCL1, which exhibits high sensitivity and specificity and with no cross-reaction with other parasitic diseases. High production of this enzyme can be obtained by purification of


MicroRNAs (miRNAs) are a class of non-coding RNA, which have recently been shown to have a wide variety of regulatory functions in relation to gene expression. Since their identification nearly 20 years ago, miRNAs have been found to play an important role in cancer, including in neurofibromatosis type 1 (NF1)-associated tumours. NF1 is the most commonly inherited tumour predisposition syndrome and can lead to malignancy via the development of malignant peripheral nerve sheath tumours (MPNSTs). Although the mechanisms by which benign neurofibromas develop into MPNSTs still remain to be elucidated, it is becoming increasingly clear that miRNAs play a key role in this process and have the potential to be used as both diagnostic and prognostic markers of tumorigenesis. They function predominantly as post-transcriptional regulators of gene expression by hybridizing to the 3993 [let7, indicat pathway , underliIn this review, the structure, function and biogenesis of miRNA molecules are discussed in reference to the recent developments that focus upon the role of miRNAs in relation to neurofibromatosis type 1.The process of miRNA biogenesis involves miRNA transcription, the transport of the miRNAs to the cytoplasm and subsequent maturation. miRNA biogenesis has been reviewed in detail by Ladomery et al.  and willThe mechanism by which mature miRNAs regulate gene expression depends upon the level of complementarity that exists between the miRNA and its target mRNA . Gene simiRNA expression profiles can be used both to categorize different types of cancer and to identify miRNA markers that can help in making prognostic predictions . Initial′UTRs of miRNA genes have been reported to be associated with cancer by virtue of their altering gene expression. SNPs may occur in miRNA biogenesis pathway genes, primary miRNAs, pre-miRNAs or mature miRNA sequences and have the potential to affect the efficiency of miRNA binding to their target sites or alternatively can create or disrupt miRNA binding sites [′UTRs with the potential to either disturb or create miRNA target interactions. They confirmed the loss-of-function and gain-of-function SNPs by luciferase assay.The potential role of miRNAs in pathology has been studied by looking not only at their single nucleotide polymorphisms (SNPs) but also at their copy number variation (CNV). Functional polymorphisms in the 3ng sites . In a reng sites  identifiDICER1) and Argonaute 2 (EIF2C2), both of which are required for miRNA biogenesis. These findings are compatible with the view that somatic miRNA copy number changes are common in cancer and could potentially account for at least a proportion of the miRNA deregulation found in many tumour types. Using bioinformatic tools, Marcinkowska et al. in 2011 [Zhang et al. in 2006  found th in 2011  have demNF1 gene located at 17q11.2 [Neurofibromatosis type 1 (NF1), a familial tumour predisposition syndrome, is characterised by the growth of benign and malignant tumours involving the peripheral and central nervous system. NF1 results from inactivating germline mutations of the  17q11.2 . Most NFNF1 gene inactivation is essential for tumour development. However, it is now known that additional molecular changes and the tumour micro-environment are associated with the progression of this type of tumour. There is currently no defined molecular signature for MPNST development. Constitutive activation of several critical cell signalling cascades also occurs in MPNSTs. Multidisciplinary collaborative efforts are clearly essential to fully decipher both the complex molecular basis of MPNST development and to define potential therapeutic targets.The process of MPNST pathogenesis is poorly understood owing to its complex histopathology and the underlying molecular mechanisms. Biallelic A general characteristic of cancer cells is uncontrolled growth and, in many cases, miRNAs can either promote or suppress this growth. Previous investigations have shown that miRNAs have key functions in the development of cancer, in relation to a range of different cellular processes including cell differentiation, developmental control, neural development, cell proliferation, apoptosis ,35 and oSeveral research groups have studied the role of miRNA in NF1-associated malignancy. Here we focus on miRNAs miR-29c, miR-34a, miR-214, miR-10b, miR204 and miR-21 which have previously been directly implicated in NF1 tumorigenesis  and suppressed cell growth (p < 0.05) while upregulating the level of PDCD4 protein. Hence, these results indicate that miR-21 is likely to play an important role in MPNST progression through its target PDCD4.In a recent study, Itani et al.  analysedNF1 mutations [MIR3652 and MIR193A at 17q11.2 encoding miR-365-2 and miR-193a, respectively) displayed no differences in the expression between the tumour types, despite miR-193a having been previously categorized as a tumour suppressor in oral squamous cell carcinoma [It has been estimated that 5% of NF1 patients possess a germline 1.4-Mb microdeletion . This reutations . Analysiutations . A recenutations  looked aarcinoma  and desparcinoma . FurtherTömböl et al.  analysed′ regulatory regions of genes is a major epigenetic mechanism. The role of aberrant DNA hypermethylation in the regulation of miRNA expression in human cancer has also been explored [DNA methylation in the 5explored . This stBoth DNA methylation and mRNA regulation can suppress gene expression and their corresponding gene product. Su et al.  demonstrin vivo. Just as one miRNA may regulate multiple targets, one target may also be regulated by a number of different miRNAs. Hence, specificity is an important factor to consider in designing a therapeutic approach.Dysregulation of miRNAs is fundamental to the pathogenesis of many cancers. miRNAs that regulate the expression of tumour suppressor genes and oncogenes are candidates for use in targeted therapies . An imprmiRNAs are increasingly being identified as useful diagnostic and prognostic markers. One example of this is in hepatocellular carcinoma (HCC) in which it has been shown that low levels of miR-26 can be indicative of a poor prognosis . These pDICER1, DROSHA and XPO5 (exportin 5), have also been shown to have therapeutic potential, at least in the case of bladder urothelial carcinoma. These genes are vital for miRNA development, and their silencing results in the inhibition of cell proliferation and apoptosis. Han et al. [DICER1, DROSHA and XPO5 could play active roles in carcinogenesis.As well as individual miRNAs being able to act as biomarkers and therapeutic targets, Han et al. , have shn et al.  showed thttp://www.prometheuslabs.com), that determines the expression levels of 48 different miRNA biomarkers in a tissue sample, from which the results are used to determine the tissue of origin of metastatic cancer.The use of miRNAs as useful biomarkers of tumorigenesis is supported by the finding that they are secreted into bodily fluids  and, henThe discovery of miRNAs nearly 20 years ago introduced us to a new regulatory mechanism enabling a better understanding of the molecular pathogenesis of cancers. Dysregulation of miRNAs is fundamental to the pathogenesis of many cancers. miRNAs have the potential to target as many as several hundred genes simultaneously making them attractive prognostic biomarkers and therapeutic targets in cancer. miRNAs are not only involved in tumour progression, but also play a role in cancer invasion, metastasis, epigenetic alterations, chemo-resistance and radio-resistance. Although research on the role of miRNAs in NF1 tumorigenesis is still in its infancy, the experimental data obtained so far indicate that a number of miRNAs may be involved in NF1 tumorigenesis, including miR-29c, miR-34a, miR-214, miR-10b, miR-204 and miR-21. However, we lack an understanding of how different miRNAs identified in NF1 tumours might interact with neurofibromin and other tumour-associated proteins and whether these miRNAs can be linked to specific cancer signalling pathways. miRNAs involved in regulating the expression of oncogenes and tumour suppressor genes are candidates for targeted therapy for NF1 tumours. Therefore, it is hoped that the study of these small RNAs will eventually make a significant difference in treating NF1, at least to delay, if not entirely eliminate, the onset of tumorigenesis. The identification not only of differentially expressed microRNAs in tumours but also their target genes is providing new avenues for therapeutic approaches. As with any new therapeutic approach, there are of course many obstacles to be overcome before miRNAs can be used in clinical practise. However, it appears to present a very promising therapeutic avenue.The authors declare that they have no competing interests.AS contributed to the compilation of the first draft, DNC and MU improved the manuscript. All authors read and approved the final manuscript.


About 4% of patients with lymphoma or leukemia suffered from rheumatologic paraneoplastic symptoms like arthralgia and about 10% of the patients with rheumatologic or neurologic clinical symptoms develop a solid cancer or hematological neoplasm. 18FDG-PET is an interesting exam to identify an underlying malignancy when a paraneoplastic syndrome is suspected; it can detect the primitive lesion and/or the metastasis lesions. The use of the 18FDG-PET can help to detect earlier hematological neoplasm in cases of paraneoplastic syndrome without a determined cause and to treat more rapidly and specifically the patient.We showed the first image of  In fact, 18FDG-PET is a molecular and metabolic imaging modality which combines the metabolic data of PET with morphological data from computed tomography CT. 18FDG-PET is an interesting exam to identify an underlying malignancy when a paraneoplastic syndrome is suspected, and it can detect the primitive lesion and/or the metastasis lesions [In rheumatology, rare cases of nonspecific inflammatory rheumatisms with arthralgias and arthritis are paraneoplastic syndrome. About 10% of the patients with rheumatologic or neurologic clinical symptoms develop a solid cancer or hematological neoplasm . Explora lesions .18FDG-PET result of a 73-year-old man with seronegative symmetric inflammatory polyarthritis. He had a history of wrist, metacarpophalangeal, and ankle and knee joints arthralgias for two years which increased for 10 months without an inflammatory biological syndrome (ESR: 5 mm at first hour and PCR: 5 mg/L). He was hospitalized because the arthritis of the ankles and knees appeared with significantly swollen and warm joints. The stiffness increased in the morning and lasted for two hours. He had no fever or fatigue but a progressive loss of 6 kilos in the last year and a half. Erythrocyte sedimentation rate was 5 mm in the first hour, C reactive protein was 5 mg/L, and blood count was normal. Rheumatoid factors, ACPA, and antinuclear antibodies were negative. Body computed tomography (CT) did not show specific paratracheal infracentimetric lymph node without tissular lesion. The 18FDG-PET result . In our case, it was a very useful exam because it pointed out hypermetabolic lesions and their sites and evoked diagnosis. 18FDG-PET can lead clinicians towards a diagnosis. The use of the 18FDG-PET can help to detect earlier hematological neoplasm in cases of paraneoplastic syndrome without a determined cause and to treat more rapidly and specifically the patient. We showed in this observation the first image of


After a gap of almost 60 years following the development of warfarin, 2 new categories of oral anticoagulant agents have been approved for clinical use – the direct thrombin inhibitors and factor Xa inhibitors. These agents promise to be more convenient to administer with fixed dosing but still have equivalent efficacy and improved bleeding risk compared to warfarin. The clinical community is looking forward to the widespread usage of these agents but there is also some apprehension regarding bleeding risks, non-availability of specific reversal strategies and lack of specific monitoring parameters. This review article will attempt to educate the reader about three representative drugs from these classes: Dabigatran, Rivaroxaban and Apixaban. We will discuss the historical perspective to the development of these drugs, available research data and pharmacology of these agents. The best strategies for monitoring and reversal of these drugs in special situations will also be touched upon.  Therapeutic anticoagulation is widely used to treat and prevent thromboembolic disorders. Anticoagulant agents not only prevent new clot formation but also facilitate intrinsic mechanisms of clot lysis by retarding existing clot progression. Effective anticoagulation has formed the basis of treatment for acute venous thromboembolic (VTE) events (deep venous thrombosis and pulmonary embolism) for a long time and reduces the mortality rate in this condition from 30% to 3-8% , 2. AntiThe introduction of heparin in the 1930s was a major breakthrough and provided the first widely available anticoagulant agent . The majOur improved knowledge of pharmacology and coagulation pathways has allowed us to develop newer anticoagulants which have shown significant promise. Beginning in the 1980s the low molecular weight heparins, enoxaparin and dalteparin being the principal agents, and then selective indirect factor Xa inhibitors like fondaparinux have been introduced [Newer oral anticoagulant agents have begun to emerge only recently and promise significant advantages over warfarin. These agents can be principally divided into two groups – direct thrombin inhibitors and oral factor Xa inhibitors. The first agent to be marketed in the first group was Ximelagatran and after 2 decades of development it was approved for use in DVT prophylaxis in 7 European countries by 2004. Despite showing significant efficacy, it never received FDA approval due to concerns about significant hepatotoxicity in a small proportion of patients. It was withdrawn from all markets in 2006 due to increasing reports of hepatotoxicity. The experience with this agent led to the development of another drug in the same class namely dabigatran. The other class of upcoming oral anticoagulants belongs to a different group of molecules that directly inhibit factor Xa. They are termed direct as they do not require antithrombin III to mediate their interaction with factor Xa. The clinically studied agents in this class include Rivaroxaban and Apixaban. The other agents in this class which have not yet completed phase 3 trials include edoxaban, otamixaban, betrixaban, darexaban, TAK 442 and YM 466.1.This review will discuss and compare the known pharmacology and available clinical data on the three oral agents which have been recently studied in widely publicized, large scale human trials and have generated great excitement. These agents include Dabigatran, Apixaban and Rivaroxaban. A single target in the coagulation cascade, fixed dosing independent of weight and generally of renal function, independence from monitoring coagulation effects and rapid onset of action mark the common benefits of these drugs. However, there are significant differences in their pharmacologic profile and approved indications. This focused review hopes to familiarize the reader with some of these issues pertaining to each drug. Their role in the interruption of the coagulation cascade is schematically displayed in Fig. et al for Boerhinger Ingelheim in 2002. It is a potent, reversible inhibitor of both free and clot-bound thrombin. The 1st clinical study in 2004 showed non-inferiority to enoxaparin in DVT prevention [rd of the drug is protein bound and thus this part is non-dialyzable. The excretion is primarily renal (80%) and half life varies from 9-16 hours depending upon age of the subject. Plasma concentration of dabigatran does not depend on liver metabolism but it is increased or decreased by inhibitors or inducers of the p-glycoprotein transporter respectively. Amiodarone, dronedarone, quinidine, ketoconazole and verapamil being p-glycoprotein inhibitors raise levels of dabigatran. Rifampin being an inducer can decrease dabigatran levels. See Table 1 for pharmacologic summary and comparison with the other new anticoagulant agents.Dabigatran was first synthesized in the lab by Norbert H. Hauel evention . It is a1The RE-NOVATE  and RE-MODEL  studies established the non-inferiority of dabigatran compared with enoxaparin at a dose of 40 mg sq once daily or 30 mg sq twice daily for VTE prophylaxis after hip and knee replacement surgeries respectively , 12. TheThe later administration of dabigatran (6-12 hours post-operatively) at a dose of 75-220 mg once daily has, however, been shown to be inferior to enoxaparin at a dose of 30 mg SQ twice daily (given after similar period post-operatively) as shown in the RE-MOBOLIZE  trial . The VTE2The RE-COVER (Dabigatran versus Warfarin in the Treatment of Acute Venous Thromboembolism) trial proved non-inferiority of dabigatran 150 mg twice daily compared to warfarin for long term anticoagulation after patient with an acute VTE event had received 5-10 days of parenteral anticoagulation with IV heparin or SQ enoxaparin [32 score = 2.1) [2.The RE-LY  trial compared dabigatran at a dose of 110-150 mg twice daily with dose adjusted warfarin in 18,113 patients with non-valvular atrial fibrillation (average CHADSe = 2.1) . At a do4No trial to date has been published assessing the use of dabigatran for use in this patient population. Animal studies show promise , 18.5Use of dabigatran in the setting of acute coronary syndrome as the anticoagulant of choice has not been evaluated. Its use to assess safety in those with a recent myocardial infarction (MI) to prevent recurrence of MI over a 6 month period was studied in the RE-DEEM  trial . In thisThe FDA approved adult dabigatran dose for non-valvular atrial fibrillation is 150 mg twice daily. The dose for renally impaired  patients is 75 mg twice daily based on pharmacokinetic modeling. The dose for CrCl <15 ml/min is not defined.The patients receiving dabigatran for post-operative prophylaxis should be administered 220 mg once daily, but dose should be restricted to 110 mg when prescribing it between 1-4 hours post-operatively. The duration of therapy for knee replacement patients is 10 days. That for hip replacement patients is 28-35 days.Apart from bleeding complications including intracranial hemorrhage, gastrointestinal hemorrhage, hematuria, dyspepsia is the major side effect of dabigatran. The relationship of dabigatran levels with aPTT  is non-linear. However at lower doses use of dabigatran does correlate with a higher aPTT and can be used to assess compliance.Thrombin time gives a fair qualitative idea of the level of anticoagulation with dabigatran. Ecarin clotting time has a linear relation with dabigatran levels but is not recommended for routine clinical use.No good antidote exists to reverse dabigatran effects in life-threatening bleeding situations like intracranial hemorrhage. Waiting for about 4 half lives (2 days) would result in elimination of most of the drug and its effect. For faster extraction of drug, hemodialysis is a viable option and can get rid of 60% of the drug in 2-3 hours. Prothrombin complex concentrates (PCC) which contain all or most of the vitamin K-dependent clotting factors have been shown to rapidly reverse dabigatran’s anticoagulant effects . Their urd is metabolized in the liver and half of this part is excreted in the urine and half in the feces. The hepatic metabolism of the drug does depend on the CYP3A4 system and makes it prone to interactions with drugs that use this pathway. Drugs such as ketoconazole (and congeners), ritonavir which inhibit CYP3A4 can cause rise in rivaroxaban levels. Similarly, inducers of this enzyme like Rifampin, carbamazepine and phenytoin can lead to a drop in rivaroxaban levels. Like dabigatran, rivaroxaban elimination also depends on the p-glycoprotein system and interactions with drugs inducing or inhibiting p-glycoprotein system are possible. See Table 1 for pharmacologic summary and comparison with the other new anticoagulant agents.Rivaroxaban is a small molecule direct inhibitor of factor Xa and is administered in its active form. It has excellent bioavailability at 80% when taken orally. It has a rapid onset of action with maximal inhibition of factor Xa noted within 1-4 hours of ingestion. About 95% of the drug is protein bound making dialysis useless to achieve rapid elimination of drug. The half life of the drug is 7-13 hours (with higher duration with increasing age). A third of the drug is excreted unchanged in the urine (as active form); the remaining 2/31The phase III RECORD ((REgulation of Coagulation in ORthopedic Surgery to Prevent Deep Venous Thrombosis and Pulmonary Embolism) studies comprised of 4 separate trials. The RECORD1 trial compared 10 mg of rivaroxaban daily (starting 6-8 hours post-operatively after THR) to 40 mg SQ of enoxaparin administered for 31-39 days . The REC2The EINSTEIN-DVT  trial compared rivaroxaban with standard therapy in patients with acute DVT but without symptomatic pulmonary embolism (PE) . Oral ri32 score of the patients was 3.5. Rivaroxaban was shown to be non-inferior to dose adjusted warfarin in terms of achieving the primary end-point. No differences in overall bleeding complications were noted between the 2 groups although significant reductions in fatal and intracranial bleeding were noted in rivaroxaban arm. See Table 2.The ROCKET-AF  trial was a double blind randomized control trial to assess the efficacy of rivaroxaban 20 mg daily in comparison to dose adjusted warfarin in patients with non-valvular atrial fibrillation . The mea4No human studies have been reported assessing the efficacy of rivaroxaban to prevent mechanical valve thrombosis, preliminary in-vitro studies have shown some promise .5Rivaroxaban has been studied to prevent cardiovascular events in a secondary prevention role after a recent acute coronary event. The recently published ATLAS ACS 2–TIMI 51  trial recruited patients admitted for an acute coronary syndrome within the last 7 days . It diviThe FDA approved rivaroxaban dose for DVT prophylaxis is 10 mg once daily for a period of 12-14 days after TKR and 35 days after THR.The FDA also approved rivaroxaban for stroke prophylaxis in atrial fibrillation on November 4, 2011 at dose of 20 mg once daily. The dose for patients with moderate renal impairment (CrCl 15-50 ml/sec) is 15 mg once daily. The drug is to be avoided for severe renal impairment (CrCl <15 ml/sec).It is also recommended that the drug be avoided in moderate to severe hepatic impairment.Bleeding related to its anticoagulant effect is the most common adverse effect. Syncope and thrombocytopenia are uncommon effects. Routine monitoring of the anticoagulant effects of rivaroxaban is not recommended. However, the tests which correlate with its anticoagulation efficacy are prothrombin time (PT) and endogenous thrombin potential (ETP). Just like dabigatran, there is no specific antidote for rivaroxaban. Waiting for the effect of the drug to be eliminated from the system is the best option when feasible. Unlike dabigatran, rivaroxaban cannot be hemodialyzed as 95% of the drug in the serum is protein bound. However, using prothrombin complex concentrates is more effective in cases of acute life threatening bleeds related to rivaroxaban. A recent study showed complete reversal of anticoagulant effects of rivaroxaban with infusion of PCC . In-vitr1 for pharmacologic summary and comparison with the other new anticoagulant agents.Apixaban is another oral direct factor Xa inhibitor which has been studied in recent clinical trials. It is administered as an active compound and has rapid onset of action with a bioavailability of 50%. The peak plasma levels are achieved about 3 hours after oral administration. The drug is 87% protein bound. The half life is 8-15 hours and 75% elimination is via the liver after metabolism using the CYP 3A4 pathway. 25% of the metabolized drug is excreted renally. Inhibition of both the CYP 3A4 and p-glycoprotein can increase drug levels. See Table 1The ADVANCE-1  trial compared apixaban 2.5 mg twice daily to enoxaparin 30 mg sq twice daily for VTE prophylaxis in patients undergoing TKR. These drugs were initiated 12-24 hours after surgery and were administered for 10-14 days . There w2http://clinicaltrials.gov/;NCT00643201).The role of apixaban in the treatment of an acute VTE event is being investigated under the ongoing AMPLIFY trial (32 score = 2) [The AVERROS  trial was a randomized control study which compared 5 mg twice daily of apixaban with 81-324 mg of aspirin in patients with atrial fibrillation not deemed to be candidates for Warfarin (mean CHADSore = 2) . The pri2 score of patients in this trial was 2.1. 18,210 patients received apixaban 5 mg twice daily or dose adjusted warfarin (for an INR =2-3) over an average follow up period of 1.8 years. This study showed a superiority of apixaban over warfarin in terms of reaching the primary end-point as defined by ischemic or hemorrhagic stroke or systemic embolism . The risk of bleeding or hemorrhagic stroke was also lower in the apixaban group . The all-cause mortality in the apixaban group was 3.52% vs. 3.94 % in the warfarin group (p=0.047). See Table 2.The ARISTOTLE  trial compared apixaban with warfarin in patients with atrial fibrillation or flutter deemed to be high risk for stroke . The ave4We are not aware of any published evidence of the use of apixaban in this setting at the time of writing of this manuscript.5The APPRAISE (Apixaban for Prevention of Acute Ischemic and Safety Events) was a phase II study compared apixaban with placebo in patients with recent non-ST-elevation MI or with ST-elevation MI when administered for 6 months after the event in addition to current standard medical therapy . This trth 2011, the FDA conferred on apixaban priority-review designation to expedite its review and potential approval. The drug doses are not yet formalized for this agent. The drug should be used with caution in patients with severe renal failure (CrCl 15-0 cc/min) and be avoided in those with end stage renal disease (CrCl < 15 cc/min).On November 29A package insert for apixaban is not yet available; most of the information regarding adverse effects can be estimated from the reported complications in the trials that have been conducted using apixaban. Bleeding seems to be the most common complication of apixaban use , 36. TheNewer anticoagulants are non-inferior to warfarin in terms of systemic embolism and perhaps dabigatran 150 mg bid is marginally better. However, if a given patient’s INR is therapeutic most of the time, is it reasonable to switch to one of the newer agent? Taking all three trials in to consideration, the average time INR was therapeutic is in the 60% range. If the INRs have been therapeutic 70% of the time, will the non-inferiority margin be breached? Our own perspective is that if the INRs are therapeutic most of the time, then it is reasonable to continue warfarin. In patients with anticipated surgeries and bleeding issues, it is better to stay with warfarin. Which one of the newer agent is preferable? Based on available data, if the patients CHADS2 score is 3 or greater, it would be preferable to use rivaroxaban as it was studied in high risk population. Apixaban, on sub group analysis was less efficacious in younger patients (<65 Yrs) and Dabigatran related bleeding risk substantially increases with advancing age (>75 yrs). Potential for reversibility is better for factor Xa inhbitors (rivaroxaban and apixaban) than direct thrombin inhibitor (dabigatran).These factors may influence the choice of individual agent in a given patient.Lastly, should rivaroxaban be given twice daily? Post market analysis may shed light on this issue.3.The newer oral anticoagulant agents have justifiably created significant excitement in the medical community. Major bleeding episodes appear to be less frequent in comparison to warfarin. Their use in stroke prophylaxis in atrial fibrillation for qualifying patients seems likely to become popular given the strong evidence. Their role in secondary prevention of MI after an acute coronary event is not justifiable at this point of time. The relative benefit of these agents in comparison to warfarin in terms of number needed to treat is displayed in Table There do remain issues with their use. Monitoring for these agents is not perfected; even though they do not require routine monitoring; this may be of importance in critical situations like assessing bleeding risk prior to emergency surgery and during major life threatening bleeds. Reversal of these agents in these setting also has not been studied adequately and may make clinicians apprehensive in using them especially in patients with high bleeding risk. In summary, the introduction of these agents has added to the armamentarium of the physician important new classes of drugs. These pharmaceutical agents have potential benefits and significant risks. Their use requires familiarity with these drugs and good clinical judgment.


Distal radius fracture is a common injury and may result in substantial dysfunction and pain. The purpose was to investigate the relationship between distal radius fracture malunion and arm-related disability.The prospective population-based cohort study included 143 consecutive patients above 18 years with an acute distal radius fracture treated with closed reduction and either cast (55 patients) or external and/or percutaneous pin fixation (88 patients). The patients were evaluated with the disabilities of the arm, shoulder and hand (DASH) questionnaire at baseline (concerning disabilities before fracture) and one year after fracture. The 1-year follow-up included the SF-12 health status questionnaire and clinical and radiographic examinations. Patients were classified into three hypothesized severity categories based on fracture malunion; no malunion, malunion involving either dorsal tilt (>10 degrees) or ulnar variance (≥1 mm), and combined malunion involving both dorsal tilt and ulnar variance. Multivariate regression analyses were performed to determine the relationship between the 1-year DASH score and malunion and the relative risk (RR) of obtaining DASH score ≥15 and the number needed to harm (NNH) were calculated.The mean DASH score at one year after fracture was significantly higher by a minimum of 10 points with each malunion severity category. The RR for persistent disability was 2.5 if the fracture healed with malunion involving either dorsal tilt or ulnar variance and 3.7 if the fracture healed with combined malunion. The NNH was 2.5 (95% CI 1.8-5.4). Malunion had a statistically significant relationship with worse SF-12 score  and grip strength.Malunion after distal radius fracture was associated with higher arm-related disability regardless of age. Patients with a distal radius fracture generally recover within six months after fracture, although a minority of patients experience prolonged functional impairment and pain . CurrentWe conducted a prospective population-based cohort study of patients with distal radius fracture to investigate the relationship between fracture malunion and arm-related disability at 1 year after fracture.From January 2001 through March 2002 we prospectively enrolled consecutive patients with distal radius fracture at one emergency department in northeastern Scania health district in southern Sweden. The orthopedic department is the only facility in that health district where closed or open reduction of distal radius fractures is performed and all patients were treated at this facility. The inclusion criteria for the present study were acute fracture of the distal radius treated with closed reduction and cast or with closed reduction and external fixation or percutanoues pin fixation and patient age above 18 years. The exclusion criteria were residence outside the region at the time of fracture according to the national population register, severe medical illness or cognitive disorder precluding participation in the follow-up examination, unwillingness to participate, treatment with open reduction and internal fixation (ORIF), and death within one year from fracture date. The study was approved by the Regional Ethical Review Board and informed consent was obtained from each patient.Data concerning type of trauma were obtained. Fall at the same level from an upright position was classified as moderate trauma and all other types of trauma  were classified as severe trauma.Nine patients were excluded because of severe medical illness or cognitive disorder, nine patients declined to participate, 10 patients were excluded because of treatment with ORIF and six patients died within one year from fracture date. A total of 143 patients (110 women and 33 men), mean age 65 years  participated in the study  and manipulation under fluoroscopy. Patients requiring surgical fixation were treated at the operating room under regional or general anesthesia. Radiographic examination was done one week after initial treatment. Fractures that were judged to have redisplaced at one week were considered for closed re-reduction and external fixation. The decision to perform secondary external fixation was made by the treating orthopedic surgeon after discussion with the patient.The patients completed the disabilities of the arm, shoulder and hand (DASH) questionnaire, sent by mail, at one week (questionnaire inquired about disabilities before the fracture) and at one year after the fracture. The DASH questionnaire (30 items measuring disability and symptoms related to the upper extremity) is scored from 0 to 100 with higher score indicating higher disability . The valThe patients also completed the SF-12 health status questionnaire during the 1-year clinical examination at the hospital. The SF-12 questionnaire has 12 items and produces a physical component score (PCS) and a mental component score (MCS), each normalized to mean of 50 and standard deviation of 10 compared to the general US population, with higher score indicating better quality of life .® Hydraulic Hand Dynamometer, Sammons Preston, Inc. Bolingbrook, IL, USA and range of motion  with a goniometer. The measurements were done on both arms.At one year after the fracture the patients were examined by a physiotherapist (MH) who measured grip strength with the JamarStandard posteroanterior and lateral radiographs were obtained before treatment (to verify the diagnosis) and at one week after treatment. At one year after fracture radiographic examination of both wrists was done. At the conclusion of the study an experienced radiologist (MP) with no knowledge of the patient's DASH responses classified the fractures according to the AO system and precisely measured dorsal tilt (degrees), ulnar variance (mm) with 1-mm intervals, radial inclination (degrees) and articular step-off (mm). Dorsal tilt was measured on the lateral view as the angle between a line connecting the dorsal and volar lips of the distal radius and a line perpendicular to the central axis of the radius, as described by Goldfarb et al . Ulnar vThe DASH was missing both at baseline and at one year in 40 patients (28%) Figure . CompareTwenty-five patients 17%) did not attend clinical examination. The baseline radiographs of nine patients and the 1-year radiographs of 14 patients were missing. The 1-year nonrespondents (46 patients with no DASH and/or radiographs at one year) did not differ significantly from the 1-year respondents (97 patients) with regard to age, sex, treatment method or fracture type  and medians were calculated. In all analyses 95% CI were calculated when appropriate. The primary outcome was the DASH score at one year after fracture. To assess the relationship between the radiographic variables measured and disability, we performed multiple linear regression analyses with the DASH score at one year as a dependent continuous variable and each of the variables dorsal tilt, ulnar variance and radial inclination at one year as independent continuous variable, adjusting for age, sex, fracture AO type, treatment method and the corresponding radiographic variable in the uninjured wrist. Of the radiographic variables, dorsal tilt and ulnar variance were found to have statistically significant effect on the DASH score Table , whereasFor our primary outcome variable (1-year DASH score) we used analysis of covariance (ANCOVA) to calculate the mean differences in DASH scores between patients with dorsal tilt ≤ 10° versus >10° and patients with ulnar variance ≤ 0 mm versus ≥1 mm as well as between the three malunion severity categories adjusting for age, sex, and treatment method. In addition, we performed a fixed-time Cox regression analysis (robust variance) with the 1-year DASH score as the dichotomized (≥15 and <15) dependent variable and age, sex, fracture AO type, treatment method, and malunion severity category as covariates. We calcFor the analysis of the secondary outcomes, we used ANCOVA to determine the association between the SF-12 scores at one year and the malunion severity category adjusting for age, sex, and treatment method . Similarly, the relationships between grip strength and forearm supination and the malunion severity category were analyzed with ANCOVA adjusting for age, sex, treatment method, dominance of the injured hand, and contralateral grip strength or supination . Additional analyses with adjustment also for radiographic parameters in the uninjured wrist did not change the results. All statistical tests were 2-sided and a p-value below 0.05 was considered to indicate statistical significance.The mean DASH score showed worsening from baseline (before injury) to one year and was similar in both treatment groups Table . The extThe mean DASH score  was significantly worse in patients with dorsal tilt >10° than those with dorsal tilt ≤10°  and in patients with ulnar variance of ≥ 1 mm than in patients with ulnar variance of ≤ 0 mm   compared to patients with no malunion  was 39.3 (SD 9), for those with malunion involving either dorsal tilt or ulnar variance (n = 65) was 45.2 (SD 10) and for those with no malunion (n = 34) was 47.9 (SD 10); the mean differences  were 6.0  and 6.8 , respectively. There were no differences in mean SF-12 MCS scores according to severity category of malunion (data not shown).About 28% of the fractures were intraarticular according to the AO fracture classification (two type B and 38 type C fractures). Among patients that were treated with closed reduction and cast, 18% of the fractures were AO fracture type C, in comparison with 32% among the patients that were treated with external fixation and/or percutaneous pinning. Intra-articular step-off at one year was present in nine of the 97 1-year respondents (5 patients had a 0.5-mm step-off and four patients had a 1-mm step-off); their mean 1-year DASH score was 22 .Of the 32 1-year nonrespondents who had attended radiographic examination, but had not completed the DASH questionnaire, eight (25%) had no malunion, 19 (59%) had malunion involving either dorsal tilt or ulnar variance, and five (16%) had a combined malunion (p = 0.66 compared with the respondents). The mean DASH score for the six patients who did not attend the 1-year radiographic examination was 13.3 (SD 15).The mean grip strength for patients with combined malunion (n = 21) was 19.2 (SD 7) kg, for those with malunion involving either dorsal tilt or ulnar variance (n = 63) was 24.7 (SD 11) kg, and for those with no malunion (n = 33) was 33.2 (SD 16) kg; the adjusted mean differences were 3.3  and 5.6 , respectively. In a similar analysis no statistically significant differences in supination were found.Complications were recorded in 47 of the 143 patients. Superficial pin tract infection requiring antibiotics occurred in 32 of the 88 patients in the external fixation and percutaneous pinning group. Osteomyelitis was diagnosed in one patient with external fixation. Carpal tunnel syndrome occurred in 10 patients (six patients in the cast group), of whom seven were treated with carpal tunnel release (two in the cast group) and three with wrist splint. Rupture of the extensor pollicis longus tendon occurred in one patient in each treatment group (one was treated with tendon transfer and one declined surgery). One patient in the external fixation and percutaneous pinning group developed chronic regional pain syndrome. One patient in the cast group developed a thumb adduction contracture that recovered with physiotherapy.Our data suggest that after a distal radius fracture in adults there is a statistically significant relationship between radiological appearance at one year and patient-reported outcomes measured with the DASH questionnaire. We found that fracture malunion, defined as dorsal tilt >10° and/or ulnar variance ≥1 mm, was associated with higher disability. The mean DASH score increased by a minimum of 10 points with increased malunion severity category; a magnitude of change shown to be clinically important ,12. The In some previous studies no association was found between radiographic results and various outcome measures ,25. SoueAlthough our study was not primarily designed to compare treatment methods it showed that the group treated with closed reduction and external fixation or percutaneous pinning had more severe fracture displacement at the initial postfracture radiographs, but achieved better radiological position at one year than the cast group. Accordingly, surgical fixation was more effective than cast in maintaining fracture position. However, fixation was more effective in restoring dorsal tilt than ulnar variance, which may partly explain that the unadjusted mean 1-year DASH score was similar in both treatment groups. The DASH score at baseline showed minimal disability, but had worsened in both groups at one year. There was also a difference in anesthetic block between the two treatment groups, because unlike regional anesthesia hematoma block does not provide muscle relaxation. Comparison of the two groups should be interpreted cautiously because the surgeons selected the treatment based on severity of the fracture. Therefore, the type of treatment was adjusted for in all analyses. The purpose of the study was not to compare the effectiveness of the treatments, but to assess the relationship between malunion and disability.It is commonly believed that fracture of the distal radius causes more disability in young adults, assumed to have higher physical demands on the wrists, than in the elderly believed to better tolerate deformity. Fracture malunion has been shown to associate with higher disability among young and middle-aged adults in several studies ,26,27. IIt is generally accepted that treatment of a distal radius fracture should aim at achieving the best possible anatomical reduction in the young active patient and at minimizing the interventions in the low-demand elderly patient with multiple comorbidities . HoweverOur study has limitations. The response rate to the DASH questionnaire was about 70%. However, the patients who failed to respond did not differ substantially from the patients who responded, which suggests that the potential influence of the loss to follow-up is not likely to be substantial. The reasons why patients did not attend follow-up are unknown. In the 1-year analyses, we used a cut-off for the DASH score of 15 to indicate higher disability. Because only a small proportion of the patients achieved 1-year scores that were as low as the baseline scores the use of a higher cut-off was justified. The US population norms for the DASH have been estimated as a mean score of 10 and standard deviation of 15 for the whole population above 18 years of age; with the mean score being higher in women than in men and gradually increasing in successive age groups . We therAnother limitation is that the DASH score may reflect disability related to upper extremity disorders other than the wrist. However, this should not differ with the severity category of malunion. We included both extra- and intra-articular fractures which could be considered a disadvantage. However, patients with an intra-articular step-off were few and their mean DASH score was only slightly higher than that for the whole patient population. Thus, it would not have a substantial effect on the results. Other factors, such as articular gap that may potentially affect disability, were not recorded, but similar to articular step-off it does not seem to be a factor that would have substantially affected the results. We did not screen for previous injuries to the contralateral wrist, the radiographic variables of which were adjusted for in our multivariate analyses. However, the mean and median values for the radiographic variables of the uninjured wrist were within normal range, suggesting that the prevalence of prior contralateral wrist fracture ought to be low and would not substantially affect the results.We did not use a specific wrist score in this study. The DASH score has been shown to be a reliable and valid evaluation tool of outcome after distal radius fracture . Correlawith fracture), we believe that this cannot be a substantial problem since baseline DASH in our data was very low. Since the DASH questionnaire is self-administered uncertainty regarding accurate completion of the questionnaire is a potential problem. The DASH has however been widely used as a mailed questionnaire. Questionnaires with more than three unanswered questions were excluded.An advantage of our study is that we recorded baseline DASH score as a measure of disability before fracture. This reduces the risk of bias in cases of comorbidity. The DASH score at baseline relied on patient recall, as the questionnaires were sent to the patients within one week of fracture; a procedure previously reported . AlthougWe concluded that after a displaced extraarticular or intraarticular distal radius fracture, treated with closed reduction and cast or with external or percutaneous pin fixation, malunion in terms of a dorsal tilt exceeding 10 degrees and/or a positive ulnar variance was associated with higher arm-related disability at one year after injury, regardless of patient age or gender.The authors declare they have no competing interests.EB contributed to study conception, analysis and interpretation of data and drafting of the manuscript; MH and MP participated in acquisition of data, PW participated in analysis and interpretation of data, LD participated in critical revision of the manuscript; IA participated in study conception and design and critical revision of 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/1471-2474/12/9/prepub


In particular, the experiments have been conducted on nanoparticles of different nature, what mainly explains the observed discrepancies. This is a reply to  The various physicochemical features of nanoparticles are now well known to lead to different interactions with biological systems and induce various toxicities . We used2 NPs on Caco-2 cells and they conclude as follows:” Results of this investigation support our hypothesis that TiO2, although not toxic, has previously undescribed non-lethal effects on microvilli and intracellular-free calcium”.• In their study, Koeneman et al. observed adsorption of unmodified TiO2 NPs.• Fisichella et al. do not observe any internalization, nor any cytotoxicity nor genomics effects in Caco-2 cells after exposure to surface-treated TiO2 NPs, Koeneman et al. observed that 29.6% of TiO2 NPs were absorbed on the cell surface, with 0.4% of the cells occupied by NPs . Their SEM image  indicated that microvilli are damaged in the presence of 1000 μg/mL of unmodified TiO2 Nps, and “no longer stood erect” at 10 μg/mL due to NPs absorption onto cell surface. We considered that our results were consistent with those of Koeneman, regarding the lack of toxicity observed in their study for similar doses (10 μg/mL) of TiO2 NPs in identical cells.In their study, after 24h exposure to 100 μg/ mL of unmodified TiO2 core coated with aluminum hydroxide and PDMS layers), 90% of the organic layer is desorbed while the aluminum hydroxide layer persists at the surface of the nano-TiO2 core. Faust et al. invoke a possible degradation of the aluminum layer, seen by others [27Al NMR spectra of the T-LiteSF before and after alteration [2 is intact after alteration. The persistence of this aluminum hydroxide layer is also confirmed by chemical analysis (ICP-AES measurements). We demonstrated [2 NPs, degraded or not, while unmodified TiO2 NPs have photocatalytic activity. The ability of unmodified TiO2 NPs to generate superoxides likely explains the adverse effects  observed by our colleagues. On the contrary, the protective effect of the remaining aluminum hydroxide layer explains the absence of toxicity described in Fisichella et al.In our work, after the ‘environmental’ or ‘acidic’ alteration of the T-liteSF  displaying the same features of microvilli. Faust et al. claim that our TEM images: "do not resemble Caco-2 images and show a significant effect of their NP treatment” because “normal Caco-2 epithelium exhibit cells with polarized cytoplasm containing many electrondense organelles and cytoplasmic granules with a well-ordered array of microvilli at the cellular apex”.Faust et al. do not have the same interpretation of our SEM pictures of Caco-2 cells ; they observed possible distorted microvilli as a proof of harmful effects of our surface-treated TiOThe absence of well-ordered microvilli is simply due to the cutting method, which involved a parallel cut to the bottom of the Petri dish in order to collect larger fragments, and not transversally as was done by Koeneman et al. . This isTo conclude, these two studies do not lead to conflicting data, but are complementary studies, based on different nanoparticles, showing the importance of taking into account the surface properties of the NPs in toxicological studies.Our colleagues will certainly agree with us that these data underscore the need to further examine the toxicological effects, not only of unmodified nanoparticles , but of surface-treated nanoparticles used in engineered nanomaterials. At each stage of the lifecycle, from production to the end of life, the surface properties of the nanoparticles will be different leading to different toxicological effects.We would like to emphasize the importance of evaluating the toxicity of engineered nanomaterials throughout their life cycle in order to design them more safely in the future.The authors declare that they have no competing interests.All authors read and approved the final manuscript.