The rise of social media proved to be a fertile ground for the expansion of the acquired immune deficiency syndrome (AIDS)-denialist movement (in the form of online communities). While there is substantial literature devoted to disproving AIDS-denialist views, there is a lack of studies exploring AIDS-denialists online communities that interact with an external environment.We explored three research areas: (1) reasons for newcomers to come to an AIDS-denialist community, (2) the patterns of interactions of the community with the newcomers, and (3) rhetorical strategies that denialists use for persuasion in the veracity of their views.We studied the largest AIDS-denialist community on one of the most popular social networking services in Russia. We used netnography as a method for collecting data for qualitative analysis and observed the community for 9 months (at least 2-3 times a week). While doing netnography, we periodically downloaded community discussions. In total, we downloaded 4821 posts and comments for analysis. Grounded theory approach was used for data analysis.Most users came to the community for the following reasons: their stories did not fit the unitary picture of AIDS disease progression translated by popular medical discourse, health problems, concern about HIV-positive tests, and desire to dissuade community members from false AIDS beliefs. On the basis of strength in AIDS-denialist beliefs, we constructed a typology of the newcomers consisting of three ideal-typical groups: (1) convinced: those who already had become denialists before coming to the group, (2) doubters: those who were undecided about the truth of either human immunodeficiency virus (HIV) science theory or AIDS-denialist theory, and (3) orthodox: those who openly held HIV science views. Reception of a newcomer mainly depended on the newcomer’s belief status. Reception was very warm for the convinced, cold or slightly hostile for the doubters, and extremely hostile or derisive for the orthodox. We identified seven main rhetorical strategies of persuasion used by the denialists on the “undecided”.Contrary to the widespread public health depiction of AIDS denialists as totally irrational, our study suggests that some of those who become AIDS denialists have sufficiently reasonable grounds to suspect that “something is wrong” with scientific theory, because their personal experience contradicts the unitary picture of AIDS disease progression. Odd and inexplicable practices of some AIDS centers only fuel these people’s suspicions. We can conclude that public health practitioners’ practices may play a role in generating AIDS-denialist sentiments. In interactions with the newcomers, the experienced community members highlighted the importance of personal autonomy and freedom of choice in decision making consistent with the consumerist ideology of health care. The study findings suggest that health care workers should change a one-size-fits-all mode of counseling for a more complex and patient-tailored approach, allowing for diversity of disease progression scenarios and scientific uncertainty. The rising role of new social media in the field of health can adequately be described as a “double-edged sword”. On one hand, new social media can deliver patients empowerment in doctor-patient relationships and be a medium of new evidence-based eHealth interventions and a platform for patient online support communities where they can share useful practical experience on coping with a chronic disease. In short, social media can help create a new type of patient, the ePatient, who is “equipped, enabled, empowered, and engaged in their health and health care decisions” . In contmovement and not just a system of views ichman’s . A surveichman’s  showed tichman’s  for reviichman’s ,24-26.The rise and global penetration of the Internet has opened a large window of opportunities for AIDS denialists, who quickly jumped on the bandwagon. As the pro-denialist Group for the Scientific Reappraisal of the HIV/AIDS Hypothesis (“Reappraising AIDS”) wrote on its website: “Thanks to the ascendance of the Internet, we are now able to reinvigorate our informational campaign” (quoted in ). The woAlthough there are no reliable data on the influence of the AIDS-denialist movement in Russia and FSU countries (either online or offline), Russian PLWH community leaders both in public talks and in informal talks with the current project team members admit that the AIDS-denialist movement is on the rise and that proliferation of social networking services (SNS) in FSU countries contributes to its growing influence. As a leader of one of the most prominent PLWH communities in Russia  put it, “we are losing the battle [with AIDS denialists] on the Internet”.To summarize, the growing presence of the AIDS-denialist movement on SNS presents a serious public health threat, which contributes to higher morbidity and mortality from AIDS and HIV-related diseases, and further spread of HIV among the populace. All this warrants research of the AIDS-denialist movement on the Internet in general, and on the social networking services in particular. To our knowledge, this is the first study of its kind.This is an explorative study of the most numerous AIDS-denialist online communities on one of the most popular social networking services in Russia . As the spread of denialists’ views and the recruitment of new members into the movement are particularly challenging for public health, we have decided to (1) examine the reasons people come to the group, (2) analyze how the community deals with the newcomers, and (3) describe rhetorical strategies employed by deniers for persuasion in the veracity of their views. Gaining such knowledge could be of significant value for designing Internet interventions directed at counteracting the influence of AIDS denialists on the Internet.We have chosen an SNS group (like Facebook groups) with a manifestly AIDS-denialist name, which is open for everybody who is willing to join. By community members, we can mean only those who have formally signed up for the SNS group: in the broadest “actionist” sense, those who participate in its activities regardless of formal belonging to the group; and finally, in the most restrictive sense, only those who formally belong to the community and also participate in its activities. We used the third definition when we drew a map of friendship in the group and the second when we calculated some statistics on activities in the community and for a qualitative analysis of the community posts and comments.The group is highly visible to people who seek information about HIV and AIDS. Thus, searching the word “HIV” in Russian (at the moment of this paper’s submission) using Google’s search engine (the second most popular engine among Russian users—33.9% of users ) returnsDuring the project’s execution (March to November 2013), the group numbered around 13,000 members and had existed for almost 5 years, with the date of the first post being December 8, 2008. The primary group’s mission statements listed on its webpage are “saving peoples’ lives” from the “AIDS industry” and spreading the true word about the AIDS conspiracy. The group contains 21 hyperlinks, the majority of which are other AIDS-denialist groups or their websites on the Internet, other antimedicine groups such as “Vaccines kill”, and nationalistic groups. The group lists nine moderators who have played a crucial role in its functioning by heavily moderating its content. Like all groups in the studied SNS, the group contains a message board called “the wall”, the most visible discussion space that has become the main object of analysis. The “wall” is where most newcomers come and it is also the place where the most heated discussions take place—apparently because it is the best place to post for attracting attention. Besides that, the group site contains 104 documents, hundreds of videos, and 284 “themes” . Videos mostly include pro-denialist ads, various news items, or heavily criticized antidenialist materials. Most are re-posted from regular media and YouTube, some of which were made in Russian, with others dubbed by the sources from which they were borrowed. The main topics in discussion threads include “scientific” justifications of AIDS-denialist assertions, legal advice, discussion of AIDS in terms of conspiracy theory, advice on how to deal with medical institutions, advice for pregnant women, “harm and consequences of ART”, and direct advice topics such as “Don’t test for HIV!”. While antidenialist activists claim that such direct calls contradict Russia’s HIV laws, Russian legislation does not directly prohibit dissemination of false medical information.For outlining the group’s general picture, we used VKminer, a software developed in our lab that helped us map the friendships in the group. With this program, we also were able to download the content of the wall for the entire 2-year period and count the number of posts, comments, and likes for every participant in the “wall” activities.We used netnography as a method for collecting data for qualitative analysis. Netnography is “a specialized form of ethnography adapted to include the unique computer-mediated contingencies of today’s social worlds” . To put Social network analysis with NodeXL was used for mapping the group’s “quantitative portrait”, while for analysis of qualitative data we used Grounded Theory approach . We usedSome of the participants used their real names , some fearing possible stigmatization used fictional names (and informed the community members about it), and some went by nicknames. We use pseudonyms in this paper when we present community members’ quotes.Analysis has shown that 61.31%  of the group members were isolates, that is, they had no friends in the group. The majority (3425) of the non-isolated members belonged to the largest connected component, while the second largest component contained only 20 nodes. The visualization of the largest connected component and of the 700 active isolates is represented in We found that the share of active online community members was 9.39% . Only 4.32%  of group members generated content (posts or comments) while the rest, 5.07% , were only attention-givers in the form of “likes”. In sum, the community consists of a small core producing all the content and a large number of readers or potential readers, a small proportion of whom sometimes approves of what they read. Such community structure is not unique; however, it is not typical for inactive groups, where a dense core is seldom observed and the scarce activity is usually more evenly distributed among the moderately active members (unless such groups are “fanpages”). The dense core is therefore an indicator of intensive group dynamics and real communicative processes in the community.Most people came to the community for the following reasons: their stories did not fit the general AIDS disease narrative (see below), curiosity, concern about HIV-positive tests, desire to dissuade the community members from false AIDS beliefs, or to support them in their struggle for truth. Many of the newcomers were in confusion and despair because of their diagnosis. Consider the following quote from a newcomer’s post that illustrates one of the most important reasons for coming to the group that we were able to pinpoint—a contradiction of the newcomer’s life story with her vision of the disease progression:Here what I did when I was initiated into the caste of “the chosen”—I sought for anybody to talk about it openly and apart from psychological support counselor from AIDS-center I didn’t find anybody. After conversation with their psychologist I came to the conclusion I should get registered but for some reason someone inside told me: don’t rush…wait…My husband took the test and got a negative result! And from this moment the internal struggle began and soul-searching, and I would say God helped me to find you because before that I hadn’t even used [the name of SNS].OlgaFrom this quote we can see that Olga started to doubt the “official theory” after she had learned that her husband was negative despite their having unprotected sexual relations, which, as she sees it, contradicts the HIV science theory according to which positives infect negatives. Another group member tells how her viral load has decreased “by itself” and her immune status rose, interpreting it as evidence of the fallacy of the “official theory”:When I was pregnant I was diagnosed with HIV. And on the fifth month [of my pregnancy] my immune status is 350 cells – it is very little. it is thought that if less than 250, it is already AIDS. Viral load 85000...Next test: Immune status 750, viral load – 25000. That is, I did not take any medicines, and the viral load decreased by itself. According to the theory this is impossible. I asked them where did 60000 viruses go, one said “he doesn’t know”, the second that “maybe they mixed up something in the lab”…At this point I stopped coming to the AIDS-center.Thus, from community members posts (old and new), we can see that their stories or lab tests results contradict (or seem to contradict) what we call the “AIDS-metanarrative”. Though each member writes about one or two contradictions with this metanarrative, combining our findings we can construct a schematic narrative, many elements of which are widely known to the public from popular and popular medical discourses. This metanarrative can be outlined as follows.People get infected with HIV in situations of risk . Then in a certain period, their immune system (CD4 count) starts to lower and viral load starts to rise, and at a certain point their depleted immune system fails to defend them from a range of diseases and they die, unless they start taking highly active antiretroviral therapy (HAART). During the entire period of the disease progression, they are contagious and infect their sexual or injection partners. Pregnant women have a high chance of transmission of HIV to their children if they do not take antiretrovirals during pregnancy.However, there are some points that contradict or seemingly contradict this AIDS-metanarrative that we saw in the community members’ posts: (1) absence of a risk situation: “I couldn’t get it because I have never used drugs or cheated on my partner, and I’m 100% sure that he didn’t cheat on me either”, (2) nontransmission of HIV from a positive to a negative: “I live with my husband and we have unprotected sex, and still seven years later he’s negative”, (3) nontransmission of HIV through sharing injection equipment: “My friend was a junkie and he shared needles with other junkies but he never caught HIV”, (4) lowering of the viral load without treatment: “My viral load dropped despite I faked taking HAART and threw out the pills”, (5) rise of the immune status without treatment: “My CD4 count rose even though I didn’t take HAART”, and (6) death of HIV-positives despite taking HAART: “People take HAART and die nevertheless”.On the basis of strength in the AIDS-denialist beliefs, we have constructed a simple typology of the newcomers that consists of the three ideal-typical groups: (1) the convinced: those who already had become deniers before coming to the group, (2) the doubters: those who presented themselves as undecided as to the truth of either HIV science theory or AIDS-denialists theory, and also often posed uncomfortable questions that cast doubt over denialist views, and finally (3) the orthodox: those who openly held HIV science views.Reception of newcomers and the choice of a rhetorical strategy addressed to newcomers strongly depended on their presentation of self to the group. The decisive factor that determined the type of reception received by newcomers was, unsurprisingly, their “belief status” in the denialist views expressed in their post, although other factors such as confusion or self-confidence, cheerfulness, or a gloomy tone also mattered to some extent. Reaction of the group can be understood as positive, neutral, or negative depending on the comment’s sentiment to the newcomer’s post and also by the quantity of “likes” the post gets. Although we did not calculate “likes” formally for every newcomer’s post, the difference in likes between the “convinced” and the other types of newcomers is striking. While the “convinced” often got from 10-20 likes, all other newcomers got 0-3 likes. The content of the post with the highest chance of getting many likes was the “thank you” message to the community or/and expressed despisal of HAART. Consider the following post from Natalia that got 16 likes:Hey guys, thank you for your community! I got “+” on the tenth week of my pregnancy. Husband “-”!!! I thank him that he didn’t turn his back on me, we together started to figure out what’s going on, to enter into details. In AC [AIDS-Center] they prescribed inviraza, kombivir, and ritonavir. Two pills of each medicine twice a day! It’s 12 pills a day!!!!! Holy shit! Considering that even when I have a banal cold I have never taken anything. AC worked all my nerves!!! After visiting them I had a stomach pain! Having read all your posts, having watched the videos I got convinced that all this is a big swindle. I won’t go to AC ever. The health of my child and my nerve cells are more important. P.S. I flushed the pills down the drain! Thank you so much!!!But apart from likes, community members gave a verbal welcome in their comments to her post. She received emotional support and welcome messages, such as, “Welcome to the group!”, “Good luck and patience!”, and “Keep us informed about your battle with AC”. We call this strategy “rhetoric of reinforcement” as the newcomer is already convinced in AIDS-denialist views and the community only supports and reinforces the views and the feeling of community belonging.A different situation arises with the doubters. Any degree of doubt in the truthfulness of denialists tenets almost always caused an ostentatiously cold or hostile reaction. Nadia drove this point home in her answer to Jenia, a “doubter”:Jenia, I think that our group should be only for true dissidents, those, who have no qualms about their positions! And you create your own group for those, who’re neither here, nor there. And you will persuade each other, whether the virus exists. Let’s not interfere with each other. Sometimes one NEEDS necessary information—on lawyer’s advice, refusal [from medical treatment], and this information is just impossible to find in the mumbo-jumbo of those who still need to be persuaded and seek an answer to the question “whether the virus exists?”By far the most popular answer that the doubters received to their questions was advice to “read the group’s materials”:There are a lot of materials in the group. Please, understand us, we can’t answer the same questions every day, every day we are asked the same things, by familiarizing yourselves with the group materials you will understand who benefits and how, and there is a lot to benefit from.AnyaSuch refusals to answer the questions and reluctance to interact (which can be dubbed “strategy of avoiding”) met resistance from the doubters. Despite the cold reception and answer-dodging, the doubters tried to get answers to their questions by the following rhetorical devices: blaming the group, justification of their doubts, and appeal to the group’s mission. For instance, Alena blamed the group and simultaneously appealed to the group’s mission—spreading the word about HIV-conspiracy:Is it so hard to copypaste the specific links?Oh yes, it’s much easier to write a derogatory message—10000 characters long—about the lack of intelligence of the one who’s asked the question. Just be forewarned—after such hospitable reception I (and other interested people) have a right to think about you whatever we want. If you want to be heard and understood, learn tact and respect to the interlocutor. Aggression is inappropriate in preaching.The experienced group members to whom these requests and reproaches were addressed reacted in a defensive or even hostile manner. They explained their annoyance by denying allegations that they tried to “make” someone believe in anything, thereby appealing to the principle of “free choice”. Thus, Georgiy responded to Alena’s accusations: “This is not a place for preaching, and nobody makes you believe in anything. Actually, nobody owes you anything. You yourself choose what to believe in and whom to believe”. Alisa supported him:Read the stories of people in the group. There are people here that have [been diagnosed with] HIV, and they have healthy children, don’t drink tera [ART], don’t infect their wives (husbands) —don’t these facts seem to you striking? And whether believe us—healthy people or people who are dying from tera and advocate tera at that—is up to you.It is worth noting that newcomers often came to the community seeking advice, for instance, whether to take HIV test or not. Despite the experienced community members’ main advice of referral to the group materials, they also did give direct advice. It should be stressed that such advice in the overwhelming number of cases contradicted the seasoned community members’ position on freedom of choice and unwillingness to “enforce” their own point of view as this advice was clearly based on the denialist dogma. The most frequently observed advice that the newcomers received were “Don’t take HIV tests!”, “Don’t go to the AIDS-center!”, “Don’t take ART!”, “Don’t believe the HIV tests results!”, “Treat real diseases, not test results”, “Don’t succumb to stress as stress causes real diseases”, “Live on as though there was never HIV positive test, enjoy your life, you’re not sick with anything”, and “Live a healthy lifestyle and everything will be all right.”Finally, the last type of newcomer according to our typology, the orthodox, come to the group page either out of curiosity or willing to persuade the community members in the falsehood of their beliefs. As we wrote above, the overwhelming number of the posts and comments written by the orthodox were swiftly removed, but the netnography method allowed capturing interactions of the hard-core deniers with the orthodox. In this case, deniers realized “strategies of protection”, and the orthodox were subjected, as a rule, to collective ridicule and insults. Marat responded to a newcomer, who presented himself as a doctor: “Kirill, the most amazing people are those, who got their education and still continue to push this HIV/AIDS scam, or some are ready to sell their souls for the money???”Despite the experienced community members’ reluctance to interact with the newcomers who were doubters, the latter often succeeded in dragging the former into conversations and overcoming the “strategy of avoiding”. In this case, dedicated denialists exercised various rhetorical strategies in order to defend their position and simultaneously try to persuade doubters of the truthfulness of their ideas. Use of rhetorical strategies  with the doubters and unwillingness to interact with them may sound like a contradiction, but we should be aware that these strategies are addressed not only to a particular doubter but to the wider audience—all those who watch these interactions without engaging in them. . Having analyzed and combined the denialists’ arguments scattered on different discussions and disputes on the wall, we have determined the main rhetorical strategies of persuasion.Denialists present the scientific community as having no proof of HIV existence, and the evidence produced by scientists as unconvincing or fabricated. However, in the modern world it takes science to disprove science. This is why denialists engage in “selective distrust of scientific authority” , that isDenialists claim that the myth of HIV appeared as a result of a global conspiracy between a secret world government and “Big Pharma”, who enforced the acceptance of this myth first in the United States and then in all other countries.Thus, Alla wrote to Kirill, who presented himself as a doctor: “There are people here, who came to these conclusions [HIV is a myth] not on the basis of propaganda but on the basis of PERSONAL experience”. As we have shown earlier, this personal experience in many cases contradicts the dominant AIDS-metanarrative. This strategy is similar to the one described by Nattrass—the use of “living icons”   is used, as well as an abundance of highly technical jargon or as Kalichman calls it “technobabble” . KalichmWe would like to conclude this section by describing the doubters’ reactions to rhetorical strategies of persuasion directed to them. In most cases, they remained undecided as illustrated by the following quote from newcomer-doubter Semen: “There is no point to delve into this heap of articles and video materials, as both sides have a lot of evidence”. We found only a few cases when a doubter thanked the group for clarifying the issue and dispelling doubts as to the veracity of the denialist tenets. However, we cannot conclude based on these findings that denialists’ rhetorical strategies are ineffective, after all, many experienced group members wrote that initially they themselves had had doubts that were later dispelled as they obtained deeper knowledge of denialist evidence. As to answering the question of how these rhetorical strategies affect “lurkers”, a study with a totally different design is required. We observed only a single case when after interaction with the community’s experienced members, a newcomer-doubter took a pro-scientific stance on HIV.Contrary to the widespread public health depictions of AIDS denialists as “crazy”, “delusional”, or insulated from reason by psychological “denial” , our stuWe do not try to assert that understanding the multifaceted phenomenon of why some people become denialists can be achieved purely by analyzing their rational reasons for accepting denialist views. Obviously, there are deep psychological reasons for that. A well-known psychological concept of “being in denial” about one’s illness that brings both psychological (tranquility) and practical (one does need not to embark on a complex regimen of pill-taking) benefits is certainly a major factor in many cases of the denialist views. However, in this paper we tried to shift the focus from a traditional perspective of analyzing people’s psychological traits and their proclivity to being in denial to the question of social-structural production of denialism.On a practical level, this means that in consultation with patients, practitioners should change a one-size-fits-all mode of counseling (the AIDS-metanarrative telling) to a more complex and patient-tailored approach, allowing for diversity of disease progression scenarios and open admission of scientific uncertainty on some HIV issues (when necessary) with concomitant emphasis that diversity and uncertainty do not undermine the basic principles and findings of HIV science. Elimination of the AIDS centers’ “shadow practices” could also be very helpful in building and/or sustaining trust in doctor-patients relationships and dispelling the conspiracy myth propagated by AIDS denialists.Studying interactions of the experienced community members with the newcomers, we have also seen that the former do not try to recruit the latter by any means necessary  but This is not to say that the AIDS-denialist community is not interested in recruiting new members. We saw that they provide emotional support to the “convinced” type of the newcomers. In addition, we know that, though without enthusiasm (the lack of which can probably be attributed to fatigue of the experienced members of constantly answering the same questions), denialists employ rhetorical strategies of persuasion, which target not only the doubters but undecided lurkers as well.We have also seen that members of the AIDS-denialist online community are not a homogenous group as they vary in the extent of their involvement in the group activities and in their belief status in denialist tenets. Further research is needed in order to address the issue of stratification among AIDS-denialist communities. While there is little use debating with hard-core denialists, we suggest spending time and resources on the doubters who have doubts both in the HIV science and the denialist views. Social network analysis methods could be particularly useful for determining “susceptibles” (similar to what is done for identifying susceptibles in other fields -40) withComparison of denialist rhetorical strategies of persuasion identified in this paper with rhetorical strategies of the antivaccination movement ,31,41,42Finally, we need to gain deeper insight into why some HIV-positive people become AIDS denialists. Although we have received some preliminary answers to this question based on qualitative analysis of posts in this research, more work needs to be done. In-depth biographical interviews with HIV-positive AIDS-denialist movement members would certainly shed light on this question. Understanding the factors influencing adoption of denialist views could be very useful for practical efforts to combat the spread of AIDS-denialist sentiments.


Serum IgG4 concentrations are commonly measured in clinical practice. The aim of this study was to investigate serum IgG4 concentrations in adults and their potential relationship with demographic, lifestyle, metabolic, and allergy-related factors.Serum IgG4 concentrations were measured with a commercial assay in 413 individuals  who were randomly selected from a general adult population.Median IgG4 concentration was 26.8 mg/dL. Five out of the 413 individuals (1.2%) exhibited IgG4 concentrations >135 mg/dL, and 17 out of 411 (4.1%) exhibited an IgG4/total IgG ratio >8%. Serum IgG4 concentrations were significantly higher in males than in females and decreased with age. After adjusting for age and sex, serum IgG4 concentrations were not significantly influenced by alcohol consumption, smoking or common metabolic abnormalities (obesity and the related metabolic syndrome). Serum IgG4 concentrations were not significantly correlated with serum concentrations of proinflammatory cytokines and inflammation markers. Serum IgG4 concentrations were significantly correlated with IgE concentrations. Serum IgG4 concentrations tended to be higher in atopics  than in non-atopics, particularly among atopics without respiratory symptoms. Serum IgG4 concentrations were not significantly correlated with total eosinophil blood count. Cases of IgG4-related disease were neither present at baseline nor detected after a median of 11 years of follow-up.Studies aimed at defining reference IgG4 values should consider partitioning by age and sex. Further studies are needed to confirm the potential influence of atopy status on serum IgG4 concentrations. The fourth subclass of immunoglobulin G IgG4) is the less abundant serum IgG subclass in humans . The hal is the lSerum concentrations of IgG4 have been little studied in general populations. In a study of 172 healthy adults, French and Harrison (1984) found that IgG4 concentrations were higher in males than in females . A similIn recent decades, the methods for IgG4 measurement have improved and the interest on IgG4 concentrations has increased due to the emerging concept of IgG4-related disease (IgG4-RD), which integrates a wide number of old diseases from a new perspective –11. InflDetermining the distribution of IgG4 concentrations in the general population is important for the interpretation of reference values in clinical practice. Studies on the general population may serve to explore the potential influence of common factors and/or disorders. Accordingly, the aim of this study was to evaluate serum IgG4 concentrations in a general adult population and their potential relationship with demographic, lifestyle, metabolic, and allergy-related factors.This study forms part of a survey of the general adult population from A-Estrada, a municipality in north-western Spain. Detailed descriptions of methodology and population characteristics have been reported elsewhere . BrieflyAlcohol consumption was evaluated in standard drinking units , by summ2), overweight (25–30 kg/m2), or obese (>30 kg/m2).Body mass index (BMI) was calculated as weight (in kg) divided by the square of height (in metres). Following standard criteria, individuals were classified as normal-weight  to any food or inhalant allergen . As a co, pollens , moulds , and animal danders (dog and cat) . Weals >3 mm after 15 minutes were considered positive. Histamine (10 mg/mL) and normal saline were used as positive and negative controls, respectively. A detailed description of the SPT profile in this study population has been reported elsewhere [All subjects underwent SPTs with a panel of relevant aeroallergens in the area. The panel included mites lsewhere . The pre™ test  was performed on all participants. This serum test is based on the ImmunoCAP system, and consists of a solid-phase immunoassay for specific IgE using a balanced mixture of relevant aeroallergens causing common allergic respiratory disease. Calculation of results was performed automatically, by reference to the fluorescence response obtained for patient samples compared to the response obtained for the reference serum supplied. The test gives a qualitative result, which is either positive or negative depending on the fluorescence response. When a serum sample gives a fluorescence response higher than or equal to that of the reference serum, the test is interpreted as positive and indicative of allergic sensitization. A previous description of Phadiatop™ positivity in this population and its relationship to positive SPTs has been described elsewhere [The UniCAP-Phadiatoplsewhere .The lifetime history of respiratory symptoms was investigated at baseline in all participants by means of the following questions: (a) “have you ever had a problem with sneezing or a runny or blocked nose when you did not have a cold or the flu?”, and (b) “have you ever had wheezing or whistling in the chest at any time in the past?”, thereby exploring the presence of nasal and bronchial symptoms, respectively. Subjects who answered “yes” to either of these questions were classified as symptomatic, as reported elsewhere .The clinical records of all participants were retrospectively searched for the presence of IgG4-RD at baseline. These records were reviewed again in November 2011 for new-onset immune disease and mortality causes. Two individuals were lost to follow-up. A total of 81 individuals died during the study period. Median follow-up was 137 months .™ analyser (The Binding Site) in strict accordance with the manufacturer's instructions. IgG4 antigen concentration was measured by turbidimetric methods using latex-enhanced antibodies to increase the relative light-scattering signal of the antigen-antibody reaction, increasing the sensitivity to 0.3 mg/dL. Precision studies were performed following CLSI Evaluation of Precision Performance of Clinical Chemistry Approved Guideline (CLSI Document EP5-A) over 21 working days, with two runs per day, obtaining a CV% <5.3 within run. According to the manufacturer, median serum IgG4 concentration in 30 healthy adults was 21.5 mg/dL and the 95th percentile range was 3.9–86.4 mg/dL. A threshold IgG4 concentration of 135 mg/dL is commonly accepted as a criterion for IgG4-related disease [Serum samples were stored frozen until use. The IgG4 subclasses were measured by turbidimetry using commercially available kit  in a SPAPLUSSerum concentrations of total IgG, IgA, and IgM were determined by a commercial nephelometry assay using a BN-II device , and have been reported elsewhere . The ratSerum concentrations of total IgE were determined by a commercial chemiluminescent enzyme immunoassay , as reported elsewhere . For som™), as reported elsewhere [Serum concentrations of proinflammatory cytokines  were determined by a commercial chemiluminescent enzyme immunoassay  as the dependent variable. For that purpose, the only case with undetectable concentrations was attributed an arbitrary value of 0.4 mg/dL. For covariates, age (in years) was introduced into the equation as a quantitative variable, and binary variables entered the equation as ‘1’ (‘present’ or ‘yes’) or ‘0’ (‘absent’ or ‘no’). Variables were forced to enter the equation in all models. To account for stratified sampling, a design-based analysis including compensatory weights was performed for the estimation of IgG4 levels in the overall population. Centile curves for describing the distribution of IgG4 in relation to age and gender were generated using a combined method based on the Cole-Green and Rigby-Stasinopoulos [P-values lower than 0.05 were considered statistically significant.All analyses were performed using the Stata software package version 10 , the SPSS software package , and R-package version 3.1.2. We used the Mann-Whitney test, Kruskal-Wallis test and Jonckheere-Terpstra test  for comparison of quantitative variables, and Spearman's rank test to assess correlation. Multivariate analyses were performed using linear regression, with serum IgG4 levels  . SimilarIn addition to respiratory allergy, hypersensitivity diseases in the series included two individuals with contact dermatitis, two individuals with Hymenoptera venom allergy, and one individual with beta-lactam allergy. Serum IgG4 concentrations in these 5 cases were similar to those from the overall sample .No cases of IgG4-RD were present in the clinical records at baseline. Likewise, no cases indicative of IgG4-RD were detected during follow-up. New-onset diseases of potential immune basis included thyroid disease (n = 9), psoriasis (n = 4), rheumatoid arthritis (n = 3), ulcerative colitis (n = 2), Still´s disease (n = 1), Raynaud´s phenomenon (n = 1), autoimmune thrombopenia (n = 1), sarcoidosis (n = 1), small-vessel vasculitis (n = 1), and chronic autoimmune hepatitis (n = 1). There were no differences in baseline IgG4 concentrations between individuals who developed immune disease and those who did not (data not shown); or between individuals who died during follow-up and those who did not (data not shown).Serum IgG4 concentrations were significantly correlated with those of IgG and IgA, though correlation was weak, particularly for IgA . There wThis study shows that serum concentrations of IgG4 in the adult population vary with age and sex, and are higher in males and younger individuals. These differences are independent of both lifestyle  and common metabolic abnormalities (obesity and related disorders). According to our results, median IgG4 values may be 36% higher in males than in females. This is in contrast with total IgG concentrations, which tended to be slightly higher in females than in males in the same population . Sex difSerum IgG4 concentrations were not significantly influenced by common lifestyle factors, such as alcohol consumption and smoking. This is in contrast with serum IgA and total IgG concentrations, which are respectively increased and decreased in alcohol consumers . LikewisThere were some intriguing associations between serum IgG4 concentrations and allergy traits. Serum IgG4 and IgE concentrations were significantly correlated. This is consistent with a common Th2-driven stimulus for both IgE and IgG4 ,11. SeruThe strengths of the study include its: population-based design, random sampling and wide age range of participants; use of a reliable, standard method for IgG4 determination; long-term follow-up; and attempt to reduce confounding by controlling for a number of common factors. To the best of our knowledge, there have been no previous studies of a similar nature conducted on a general population. With regard to external validity, it should be noted that all participants were Caucasians and that many cases of IgG4-RD  have been reported in Asian populations ,15,18,20The attention paid to serum IgG4 concentrations has increased in recent years due to the emerging concept of IgG4-RD. The present study was not conducted to investigate the diagnostic accuracy of serum IgG4 for the diagnosis of IgG4-RD. Furthermore, the study was not powered to detect the incidence of such a rare disorder . The diaS1 Table(XLS)Click here for additional data file.


An elevated serum IgG4 level is one of the most useful factors in the diagnosis of IgG4-related disease (IgG4-RD). In this study, we performed a meta-analysis of the published articles assessing the diagnostic accuracy of serum IgG4 concentrations for IgG4-RD. The databases of MEDLINE/PubMed, EMBASE and Web of Science were systematically searched for relevant studies. Sensitivities and specificities of serum IgG4 in each study were calculated, and the hierarchical summary receiver operating characteristic (HSROC) model with a random effects model were employed to obtain the individual and pooled estimates of sensitivities and specificities. In total, twenty-three studies comprising 6048 patients with IgG4-RD were included in the meta-analysis. The pooled sensitivity was 85% with a 95% confidence interval (CI) of 78–90%; the pooled specificity was 93% with a 95% CI of 90–95%. The HSROC curve for quantitative serum IgG4 lies closer to the upper left corner of the plot, and the area under the curve (AUC) was 0.95 , which suggested a high diagnostic accuracy of serum IgG4 for the entity of IgG4-RD. Our study suggests that serum IgG4 has high sensitivity and specificity in the diagnosis of IgG4-RD. Immunoglobulin (Ig) G4-related disease (IgG4-RD) is an entity of emerging immune-mediated diseases characterised by the infiltration of IgG4-bearing plasma cells, elevated serum IgG4 concentration, and systemic disorders present in nearly all organs with the exception of cerebral parenchyma12+ plasma cells and fibrosis), haematological examination (concentrations of serum IgG4 >135 mg/dl), and imaging modalities (narrowing of ducts and/or enlargement of organs)674911The organ-specific diagnostic criteria for patients with IgG4-RD is likely impossible to establish due to variable organ involvements and clinical symptomsHowever, many diseases other than IgG4-RD have higher levels of IgG4, and studies have suggested an unsatisfactory performance of serum IgG4 detection in the diagnosis of IgG4-RD with poor specificity and positive predictive value despite a high sensitivity and negative predictive value21369211161718Up to now, a comprehensive overview of the accuracy and precision of the serum IgG4 concentration for the diagnosis of all IgG4-RD has not been performed. We aimed to establish the diagnostic performance of the serum IgG4 concentration for IgG4-RD involving the pancreas, bile duct, salivary gland, and lacrimal gland from non-IgG4-RD and/or healthy controls.We searched the electronic databases of MEDLINE (via PubMed), EMBASE and the Web of Science from 2000 to September 2015 in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelinesProspective or retrospective case-control studies on the utility of serum IgG4 concentration in the diagnosis of IgG4-RD were deemed eligible for inclusion in the meta-analysis. The studies also met the criteria in that serum IgG4 concentration with an unambiguous cut-off value had been evaluated between IgG4-RD with a wide variety of organs involved and other diseases, as well as healthy controls. Articles with a larger sample size or more recently published articles were included when they used the same case series. Studies for which inadequate data for confirming the diagnosis of IgG4-RD and those assessing the role of IgG4 in the pathogenic mechanism were excluded. Conference or poster abstracts without sufficient clinical information or subsequent publication in full text were excluded. Studies with fewer than 10 included patients or based on animal or cell cultures were also excluded.Risk of bias and applicability were critically assessed according to the revised Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool, which has 4 key domains including patient selection, index text, reference standard, and flow and timing2 statistics, which ranged from 0 to 100% and were interpreted as representing low, medium and high inconsistency with the values of ≤25%, ≤50% and ≤75%, respectively, in accordance with the proposal of Higgins and ThompsonThe forest plot of individual and summarised sensitivity and specificity along with 95% confidence intervals (95% CIs) of the included studies were generated to graphically represent the diagnostic value of serum IgG4 in IgG4-RD. Subsequently, a hierarchical summary receiver operating characteristic (HSROC) model with a corresponding 95% confidence contour and 95% prediction contour was calculated. A bivariate random effects model following the DerSimonian-Laird method with a corresponding test of heterogeneity was used for data pooling. The heterogeneity across studies included in the meta-analysis was statistically detected using a Q test and IThe initial keyword search yielded 2071 potentially relevant studies from the databases of PubMed (n = 533), Embase (n = 708), and the Web of Science (n = 830). After 464 duplicates were discarded, 1607 articles remained, of which the title and abstract were screened for eligibility in the meta-analysis. In accordance with the predefined inclusion criteria, 1472 articles were removed, and the remaining 135 articles were deemed potentially relevant. Following the further review of the full text articles, 111 studies were excluded due to improper design for data extraction (n = 70), insufficient data for fourfold table construction (n = 38), duplicated publication (n = 2) and published in a language other than English (n = 1). Finally, 23 articles were considered as eligible and included in this meta-analysis . No disaIn total, 6048 patients in 23 studies were included in the spectrum of IgG4-RD including AIP (13 studies), MD (3 studies), IgG4-associated cholangitis (6 studies) and IgG4-RD without further classification according to the organ involved (4 studies). The study type included 10 retrospective studies, 6 prospective studies and 7 studies without reporting the study type. Ethnicity included Asian in 15 studies with 3931 patients and Caucasian in 8 studies with 2117 patients .Overall, the included studies were of moderate methodological quality according to the QUADAS-2 tool. The high risk of bias and concerns of applicability regarding patient selection were introduced because all included studies were of a randomised or case-control design. The risk of bias for the index test and reference standard remained either (1) unclear because the index test and/or reference standard were interpreted double-blindly in all included studies with the exception of one242526272 of 87.89  (p < 0.01) and 482.65 with I2 of 95.44  (p < 0.01), both of which suggested significant heterogeneity , single radial immunodiffusion (2 studies) and ELISA (1 study). Notably, the assay of serum IgG4 was not reported in 5 studies. The sensitivity and specificity of serum IgG4 concentration for the diagnosis of IgG4-RD ranged from 32% to 100% and from 59% to 100%, respectively . The pooogeneity . The diaThe diagnostic values of the studies were demonstrated in a HSROC graph in which the summary operating point represents the pooled sensitivity and specificity, as well as 95% confidence and the prediction region represents 95% CI of the pooled and individual sensitivity and specificity. The HSROC curve for quantitative serum IgG4 lies closer to the upper left corner of the HSROC plot, and the area under the curve (AUC) was 0.95 , which suggested an impressive diagnostic accuracy of serum IgG4 for the entity of IgG4-RD. Finally, the curve is symmetrical with the Z statistic of −0.58 (p = 0.564), which also indicates a high diagnostic accuracy for serum IgG4 .The subgroup analysis was performed according to study period (published before vs. after 2011), study design (designed prospectively vs. retrospectively), sample size (less than 150 vs. more than 150), ethnicity (Asian vs. Caucasian), and serum IgG4 concentration detection assay (nephelometry vs. another method). The sensitivity of serum IgG4 was higher for (1) the studies published after 2011, (2) retrospective studies performed before 2011 that used detection methods other than assays, and (3) prospective studies using the method of nephelometry, whereas specificity was significantly different between the subgroups of all variables for the diagnosis of IgG4-RD .Egger’s regression test did not reveal any publication bias arising from small-study effects p  =  0.30) . A sensi229 . A sensAccurately differentiating IgG4-RD from malignancies such as pancreatic cancer is very important to avoid unnecessary surgeries. Compared with the histopathological criteria, the detection of serum IgG4 is one of the most convenient and valuable non-invasive examinations in clinical practice for the diagnosis of IgG4-RD, especially in disease screening at an early stage. However, because the positive rate of serum IgG4 varied according to different studies, the diagnostic accuracy of this method is controversial. A previous meta-analysis published in 2009 demonstrated that the serum IgG4 is a good marker of a single disease of AIP with a pooled sensitivity ranging from 82.3% to 89.3% and a specificity of 94.6% to 95.8% according to different controlIn the last decade, numerous studies have attempted to evaluate the diagnostic value of serum IgG4 in IgG4-RD. To summarise these studies, we conducted this meta-analysis, which included 23 studies comprising a total of 6048 patients with IgG4-RD diagnosed with different criteria. The key findings of our analysis are that serum IgG4 has a very high accuracy for sensitivity (85%) and specificity (93%) in detecting IgG4-RD involving the pancreas, bile duct, salivary gland, or lacrimal gland from non-IgG4-RD involving the same organs and/or healthy controls (or both). The serum IgG4 has a higher summarised sensitivity and specificity compared with the histopathological method using the infiltration of IgG4-positive plasma cells in which the pooled sensitivity and specificity were 58.8% and 90.2%, respectivelyOur meta-analysis had several limitations. Firstly, there was inevitable heterogeneity in the meta-analysis of diagnostic accuracy due to the variability in design characteristics and the poor quality of reporting in the primary studies. The quality of the studies included in the meta-analysis was modest for our meta-analysis because ten studies were retrospective designed192324252627282933111618303529101517372425262721118Serum IgG4 is a more cost-effective, easy and a time-efficient assay that can be carried out to detect IgG4-RD. We conclude that this meta-analysis has achieved its primary objectives by demonstrating that the detection of serum IgG4 has high sensitivity and represents a specific investigative modality in the detection of IgG4-RD as an indicator. However, this does not necessarily indicate that the positive or negative serum IgG4 could be used to rule out (or confirm) a diagnosis of IgG4-RD. A diagnosis based on IgG4-RD should be made after comprehensive analysis that considers clinical symptoms, as well as histopathologic, haematological, and imaging findings. The histopathological assessment of biopsy specimens from the involved tissues remains the cornerstone in both the definite diagnosis of IgG4-RD and the exclusion of malignancies. In summary, appropriate considerations and cautious interpretations of these findings combined with other parameters  are highly recommended, and additional studies that evaluate the accuracy of IgG4 in a wider spectrum of IgG-RD are needed.How to cite this article: Xu, W.- et al. Diagnostic performance of serum IgG4 level for IgG4-related disease: a meta-analysis. Sci. Rep.6, 32035; doi: 10.1038/srep32035 (2016).


Antibiotic-sensitive and -resistant bacteria coexist in natural environments with low, if detectable, antibiotic concentrations. Except possibly around localized antibiotic sources, where resistance can provide a strong advantage, bacterial fitness is dominated by stresses unaffected by resistance to the antibiotic. How do such mixed and heterogeneous conditions influence the selective advantage or disadvantage of antibiotic resistance? Here we find that sub-inhibitory levels of tetracyclines potentiate selection for or against tetracycline resistance around localized sources of almost any toxin or stress. Furthermore, certain stresses generate alternating rings of selection for and against resistance around a localized source of the antibiotic. In these conditions, localized antibiotic sources, even at high strengths, can actually produce a net selection against resistance to the antibiotic. Our results show that interactions between the effects of an antibiotic and other stresses in inhomogeneous environments can generate pervasive, complex patterns of selection both for and against antibiotic resistance. Antibiotic concentrations are low in most natural environments, except around localized antibiotic sources. Here, Chait et al. show that sub-inhibitory antibiotic levels can interact with many other stresses to generate complex patterns of selection for and against resistance to the antibiotic. Clinical antibiotic use has led to the evolution and spread of antibiotic resistance in bacterial pathogens234567891011131415What is the net selection for or against resistance (hereafter ‘selection on resistance') in a multistress environment? Although a gene conferring resistance to a given antibiotic might provide positive or negative cross-resistance to a few other toxins or stresses21718192021681315162324252627282930313334353337Here we find that sub-inhibitory levels of doxycycline, a tetracycline antibiotic, widely potentiate selection on resistance to tetracyclines by a diverse range of antibiotic, chemical and physical stresses. A simple model of growth of sensitive and resistant strains exposed to antibiotic–stress combinations rationalizes these observations and suggests that exceptions could be rare. We also observe a striking effect of low background levels of a second antibiotic, ciprofloxacin, on the spatial growth and selection of tetracycline-sensitive and -resistant strains around a diffusing source of doxycycline. Therefore, our results indicate that, in heterogeneous multistress environments, the selective advantage of resistance to a particular stress is affected not only by the level of that stress but also by its interactions with the multiple other stresses in the environment.Escherichia coli K12 background (strain MC4100)21223337E. coli on nutrient agar containing concentration gradients of diffusing compounds (Methods section)tetA-mediated tetracycline resistance (−1 of anhydrotetracycline added uniformly to the agar). Antibiotic-potentiated selection is identified when a compound is neutrally selective in the Type I assay, but demonstrates differential inhibition relative to the background in the ‘Type II' assay, where a sub-inhibitory concentration of doxycycline is added uniformly to the agar . As controls, we use erythromycin  and ciprofloxacin  which, respectively, show selection for and against resistance, conditioned on the presence of doxycycline (Ery ∼42 μg ml−1 and MICCpr∼6 ng ml−1), but exert a strongly differential inhibition when combined with a sub-inhibitory level of doxycycline , and those that select on differences between sensitive and resistant bacteria potentiated by the presence of a low level of the antibiotic (potentiated selection), we used a previously developed assay to measure their differential inhibition of antibiotic-resistant versus -sensitive strainsstrain MC002122333ycycline . While oycycline . These mTo study the effect of a sub-inhibitory level of a tetracycline on selection for or against tetracycline resistance by other toxins, we used our differential inhibition assay to test a set of clinical antibiotics of diverse types and targets. As anticipated from the specificity of the tetracycline efflux pump expressed by the resistant strain, we found that nearly all non-tetracycline antibiotics did not, on their own, produce a zone of differential selection either for or against tetracycline resistance . Growth 3337X=0, no selection in Type I Assay), the addition of a fixed sub-MIC level of X (X=x′>0) will almost always generate differential susceptibility to Y . In the suppressive case, Y selects in favour of X-sensitivity  with ciprofloxacin generates a region of drug concentrations selective for sensitivity to tetracyclinesWhile antibiotic resistance enjoys a clear fitness advantage in clinical settings where single drugs are used in high concentrations, the selective advantage of antibiotic resistance in natural and artificial environments with much lower antibiotic concentrations is harder to quantify. Recent reports demonstrate direct selection by sub-inhibitory levels of antibiotics on adaptations to them when the drugs still comprise the dominant environmental stress3132−1 (NH4)2SO4, 13.6 g l−1 KH2PO4, 0.5 mg l−1 FeSO4·7H2O) supplemented with 0.2% glucose, 0.01% casamino acids, 1 mM MgSO4, 1.5 μM thiamine. Solid media included 2% bacto-agar (BD). In Type I Assay, expression of the regulated TetA efflux pump without growth defect was achieved by the addition of 20 ng ml−1 anhydrotetracycline. In Type II Assay, a sub-inhibitory concentration of doxycycline , induced expression of the efflux pump and reduced the growth rate of the sensitive strains  of a tetracycline-resistant and -sensitive strain pair  and Type II (+Dox) plates in impregnated paper disks , or as a 2–3 μl drop of stock solution directly deposited on the agar surface. Paraquat (100 mg ml−1), and hydrogen peroxide (3%) were deposited directly on the agar surface as a 2.5 μl droplet. 40 μl of 0.5 M EDTA, or 75 μl of 5.1 M NaCl, 2.6 M sucrose, 30 mM PEG 8000, 2.5 M sodium citrate or 1 M CaCl2 solution were loaded into agar cups formed by removing 6-mm diameter plugs from the centre of the plates. The plates are then incubated at 30 °C for 20 h and imaged in CFP and YFP channels using a custom plate imagerAliquots of overnight stationary cultures (∼106 cells of each strain per plate) and pre-incubated at 30 °C for ∼3 h. Small regions of a Type I (−Dox) and a Type II (+Dox) plate are swabbed and struck on LB agar for verification by colony counting of a roughly 1:1 strain ratio at stress exposure . The plates are then placed side-by-side and exposed from one edge, without lids, to an infrared heat lamp  at a distance of ∼1 inch from the plate, for 5 min. The plates are cooled, uncovered at room temperature for several minutes, returned to 30 °C, incubated for 20 h and imaged.Assay plates are spread with a mixed lawn of fluorescently labelled tetracycline-sensitive and -resistant strains  and pre-incubated at 37 °C for ∼3 h. A −Dox and +Dox plate are removed and a small region of each swabbed and struck on LB agar for subsequent verification of strain ratio at exposure. The plates are then placed upright, side-by-side, without lids in a Stratalinker 1800 (Stratagene) and exposed to ultraviolet as a cover is drawn across both plates simultaneously, resulting in a roughly linear gradient of ultraviolet exposure between 0 and 7 s. Plates are then returned to 37 °C, incubated for 20 h and imaged.Assay plates are spread with a mixed lawn of fluorescently labelled tetracycline-sensitive and -resistant strains  and antibiotic-free conditions, were obtained in triplicate by serial dilutions across 96-well microtiter plates . Bacterioluciferase-expressing tetracycline-resistant . Ciprofloxacin-potentiated selection by doxycycline on tetracycline resistance was tested using the same plates, supplemented uniformly with 7.5 ng ml−1 ciprofloxacin (+Cpr plates). Doxycycline hyclate (2.5 μl of 15 mg ml−1 solution in water) was spotted at the centre of each plate, and the plates incubated at room temperature for 6 h. The plates were then spread with ∼106 each of YFP- and CFP-labelled tetracycline-sensitive and -resistant assay strains .Supplementary Figures 1-11, Supplementary Note 1 and Supplementary References


IgG4-related disease (IgG4-RD) is a newly recognized disorder, characterized by massive IgG4+ lymphocyte and plasma cell infiltration, storiform fibrosis, causing enlargement, nodules or thickening of the various organs, simultaneously or metachronously. Involvement of the gastrointestinal tract is very rare and can be presented as a diffuse wall thickening or polyp or mass-like lesion. Up to now, there have been reported only a few cases of isolated gastric IgG4-RD.We present an unusual case of IgG4-RD of the stomach with involvement of the regional lymph nodes, clinically manifested as a gastric cancer with related pyloric stenosis. The patient underwent distal gastrectomy, omentectomy and lymph node dissection. The postoperative serum IgG4 level was increased. The diagnosis was confirmed by immunohistochemical study.In the most of the reported cases there was not sufficient data about the regional lymph nodal status, although the majority of the patients had been operated with presumptive diagnosis of gastric neoplasm. Our case is rare and valuable because it presents a gastric IgG4-related lesion larger than all previously reported in literature, and IgG4-related lymphadenopathy, confirmed histologically, which contributes to better knowledge of the disease. IgG4-related disease (IgG4-RD) is a newly recognized disorder, characterized by massive IgG4+ lymphocyte and plasma cell infiltration, storiform fibrosis, causing enlargement, nodules or thickening of the various organs, simultaneously or metachronously –4. OtherWe described the case of suspected gastric IgG4-RD and regional lymphadenopathy, misdiagnosed as a gastric cancer. A 62-year-old female presented with symptoms of severe weakness and fatigue. Laboratory tests revealed anemia (hemoglobin 58 g/L). Because of these the patient had been admitted to another hospital where a gastric tumor and related pyloric stenosis were found with gastroscopy and several blood transfusions were performed. The biopsy showed a chronic ulcerative lesion with suspected atypical cells in the bottom. Computer tomography (CT) revealed prepyloric irregular thickening of the gastric wall up to 15 mm which involved its entire circumference. The patient’s co-morbidities included Henoch-Schonlein purpura, allergy to analgesics, arterial hypertension and depressive disorder. Several years ago, the patient had undergone cholecystectomy and plastic of hiatal hernia. The physical examination of the abdomen did not reveal any pathological findings. Given the presumptive diagnosis of gastric cancer, the patient was hospitalized in our department for surgical treatment. A distal gastrectomy, omentectomy and lymph node dissection were performed. The diagnosis was confirmed with histological and immunohistochemical study.Grossly, there was an ulcer-infiltrative tumor formation, 8/3 cm in size, involving gastric lesser curvature, the front and posterior wall of the stomach. The tumor infiltrated the lesser omentum and penetration into the serosa was probable. The surrounding mucosa was scattered with petechial hemorrhages Fig. .Fig. 1MaMicroscopic examination of the gastric formation showed tumor-like proliferation with mixed cellular composition: predominantly CD79a + mature B-cells, organized in follicular structures and located diffusely; abundance of mature CD 138+ plasma cells and significant presence of eosinophils Fig. . There wThe postoperative period was uneventful. Serum IgG4 level measured postoperatively was 193.1 mg/dL .IgG4-RD was firstly described in patients with sclerosing cholangitis, called autoimmune pancreatitis (AIP) type I, and later observed in other organs . The comThe most gastric IgG4-RD was detected in middle- aged patients (45 to 75 years) with predominance of women, although the total number of patients is too small to reveal any meaningful data. The body of the stomach was affected mainly. The size was variable (8–56 mm in cases reported in literature) and tumor in our case was considerably larger than the other gastric lesions of IgG4-RD. In most of the cases the gastric lesion was solitary. The two patients with multiple polyps in the stomach also had AIP and autoimmune endocrinopathy, respectively. In 7 of all reported cases, there were associated autoimmune diseases. In four of these patients there was multi-organ involvement of IgG4-RD – one patient with concomitant AIP, one case – with AIP and IgG4-related sialadenitis, one patient with localization of the disease in stomach, lungs, skin and lymph nodes and our patient had lymphonodal involvement, too. The IgG4-related lymphadenopathy, as in our case, can be confirmed only by histological examination. According to criteria, classified by Sato et al. histological changes include: 1/Castleman’s disease-like morphology; 2/reactive follicular hyperplasia, 3/interfollicular plasmacytosis and immunoblastosis, 4/progressive transformation of germinal center-like, and 5/inflammatory pseudotumor-like morphology .In most of the reported cases of gastric IgG4-RD there was not sufficient data about the lymph nodal status, although they were treated surgically with presumptive diagnosis of gastric neoplasm. In our case the accurate lymph nodal dissection was performed and IgG4-related lymphadenopathy was confirmed immunohistochemically. No enlarged lymph nodes had been detected on CT and the dissection was carried out only because we had suspected oncological disease. So if we had not done it, we would have missed the involvement of lymph nodes of IgG4-RD.The first therapeutic choice for management of IgG4-RD is the steroid treatment , 3. HoweMost gastric IgG4-RD lesions are difficult to diagnose because of their rarity and it is of utmost importance to exclude neoplasm. Unnecessary surgery can be avoided if the IgG4-RD of the stomach is considered in the differential diagnosis. Our case is rare and valuable because it presents a gastric IgG4-related lesion larger than all previously reported in literature, and IgG4-related lymphadenopathy, confirmed histologically, which contributes to the better knowledge of the disease.AIP, autoimmune pancreatitis; CT, Computer tomography; DG, distal gastrectomy; ESR, endoscopic submucosal resection; F, female; H&E, Hematoxylin and Eosin; IgG4-RD, IgG4-related disease; M, male; NA, not available; WR, wedge resection


EGFR gene mutations have shown a dramatic response to gefitinib. However, drug resistance eventually emerges which limits the mean duration of response. With that in view, we examined the correlations between MET gene status as assessed by fluorescence in situ hybridization (FISH) with overall survival (OS) and progression-free survival (PFS) in adenocarcinoma patients with EGFR gene mutations who had received gefitinib therapy.Lung adenocarcinoma patients with EGFR mutation from adenocarcinoma patients who had received gefitinib. Gene copy numbers (GCNs) and amplification of MET gene before gefitinib therapy was examined by FISH. MET protein expression was also evaluated by immunohistochemistry (IHC).We evaluated 35 lung cancer samples with MET gene (red)/CEP7q (green) per cell). IHC evaluation of MET protein expression could not confirm MET high polysomy status. The Eleven patients with MET FISH positivity had significantly shorter progression-free survival (PFS) and overall survival (OS) than the 24 patients who were MET FISH-negative (PFS: p = 0.001 and OS: p = 0.03). Median PFS and OS with MET FISH-positivity were 7.6 months and 16.8 months, respectively, whereas PFS and OS with MET FISH-negativity were 15.9 months and 33.0 months, respectively. Univariate analysis revealed that MET FISH-positivity was the most significant independent factor associated with a high risk of progression and death  and 2.25 (p = 0.03), respectively).FISH assessment showed that of the 35 adenocarcinoma samples, 10 patients (29%) exhibited high polysomy (5 copies≦mean MET per cell) and 1 patient (3%) exhibited amplification (2≦MET gene may be useful in predicting shortened PFS and OS after Gefitinib treatment in lung adenocarcinoma. The correlation between MET gene status and clinical outcomes for EGFR-TKI should be further evaluated using large scale samples.Using FISH analysis to detect high polysomy and amplification of  EGFR) gene, including the deletion mutations around nucleotide residue 746–750 in exon 19 (exon 19 deletion) and also substitution of leucine with arginine at codon 858 in exon 21 (exon 21 L858R), are correlated with sensitivity to EGFR-tyrosine kinase inhibitors (EGFR-TKIs) [EGFR mutant cases shows a dramatic response to EGFR-TKIs [EGFR-mutated advanced non–small cell lung cancer (NSCLC) [EGFR gene activating mutations, the mean duration of many patients’ successful response to gefitinib is shortened as they acquire drug resistance. Mechanisms of acquired resistance to EGFR-TKI have recently been found, such as T790M secondary mutation and MET amplification [Activating mutations of the epidermal growth factor receptor  analysis contributed to poor prognosis in NSCLC patients who received surgical treatments [MET gene activation, there may still be a substantial clinical benefit for assessing MET FISH status in NSCLC patients with EGFR gene mutations especially before initiation of EGFR-TKI therapy.GFR-TKIs . Althougse stage . A receneatments . Though MET gene copy number status as assessed by FISH could predict the clinical outcome for EGFR-TKI in EGFR-mutated lung adenocarcinoma patients.In this study, we investigated if EGFR gene mutations were obtained from 35 lung adenocarcinoma patients, all of whom had received gefitinib and provided written informed consent, at Nippon Medical School Hospital between 2008 and 2010  version 1.0. This study was approved by Nippon Medical School Hospital’s Institutional Review Board. Every patient has a signature of informed consent.Thirty-five tumor specimens with EGFR mutations by the PNA-LNA PCR clamp method as reported previously [Cytologic or histologic specimens were examined for eviously .MET gene were examined by FISH. The tissue sections were then hybridized with Met (TexRed)/CEN7q (FITC) Dual Color FISH Probe. . The number of fluorescence signals was counted independently by two investigators using an Axio Vision microscope . MET GCN was determined by FISH with probes for Met (TexRed)/CEN7q (FITC) Dual Color FISH Probe. FISH positivity was estimated using the standard Colorado criteria /CEP7q (green) per cell plus high polysomy; 5 copies≦mean MET per cell) [Gene copy numbers (GCNs) and amplification of er cell) ,11-13  was calculated from the time of gefitinib therapy to time of disease progression or last disease assessment. Overall survival (OS) was calculated from the time of gefitinib therapy to patient death or last contact. Kaplan–Meier survival curves were drawn for PFS and OS and compared by log-rank test. Univariate and multivariate analyses were performed using the Cox regression model. Statistical significance was defined as p < 0.05 for each analysis. All statistical analyses were carried out using Stat Flex version 7 .MET gene activation as estimated by FISH analysis. High polysomy was observed in 10 patients (29%) and amplification was in 1 patient (3%) of 11 MET FISH-positive cases . Exon 19 deletion was not detected at gefitinib failure in one case (case 2). Four cases were also observed to be MET FISH-positive at the failure of gefitinib treatment . One case with MET amplification before initiation of therapy was also found to harbor amplification during relapse (case 1). At gefitinib failure, coexistence of T790M and MET high polysomy of the MET gene were observed in one case (case 3). MET gene status was found to be not related to clinicopathological factors (Table Eleven of 35 patients 31%) had % had METpatients 9% and amMET amplification showed high MET protein expression (case 1), and correspondingly, no positive MET staining was observed in the 4 patients without high polysomy . However, only one of four cases with high polysomy showed moderate level of MET expression (case 8). One limitation of our study was that the number of samples was small. Thus, analysis of MET protein expression by IHC could not confirm MET high polysomy status.Next, we assessed MET protein expression levels in 9 available tumor sections by IHC and evaluated the relationship between the expression of MET and GCNs Table  Figure D-F. One EGFR gene mutation subtype was not found to be associated with sensitivity to gefitinib. Median PFS of patients harboring the L858R and exon 19 deletion mutations was 14.6 months and 13.1 months, respectively (p =0.78)  Figure A. Median) Figure B.Figure MET gene status    and 2.25 (p = 0.03), respectively (Table Responses to gefitinib were not significantly different according to us Table . However) Figure A. One caMET status of 35 lung adenocarcinoma patients with EGFR gene mutations who received gefitinib therapy. MET gene amplification before EGFR-TKI therapy was observed by high-throughput FISH analysis [MET gene amplification can become major clones after EGFR-TKI treatment and upon accumulation of secondary genetic alterations, such as the T790M mutation. In our analysis, MET FISH-positivity was also detected after continued exposure to gefitinib. Of course MET status is only one of several molecular mechanisms that account for resistance to TKIs, and was the only one we investigated in the 35 cases. Other mechanisms could lead to acquiring drug resistance. For example, it is possible that many more of the relapsed, non-tested, cases carried T790M mutations and that this was greater factor shortening the survival rate in the few MET positives cases.In this study, we evaluated by FISH analyses, the analysis . This fiMET FISH status and EGFR mutation had shorter disease-free survival (DFS) as well as OS after resection [MET FISH status and clinical outcomes of treatment with EGFR-TKIs has not been evaluated in previous reports [MET gene status. Nevertheless, MET FISH-positive patients revealed not only significantly shorter PFS but also OS from the beginning of gefitinib therapy as compared to MET FISH-negative patients. Previous reports demonstrated that gain of MET GCNs may be related to the elevation of MET protein expression and its phosphorylation [MET gene by FISH may be useful for predicting short PFS and OS after gefitinib treatment in Lung Adenocarcinoma with EGFR mutation.A recent report demonstrated that lung adenocarcinoma cases with a co-existence of positive esection . However reports ,16. Our rylation . In thisEGFR mutation with MET overexpression demonstrated that monotherapy targeting either EGFR or MET did not show tumor regression [EGFR and MET activation [Transgenic mouse models for lung cancer that express gression . In conttivation . Demonsttivation . Howevertivation . In lighKRAS mutations, although this study did not meet its primary end point [MET FISH-positive patients with EGFR mutations.Yet another phase II study with combination therapy in previously-treated NSCLC patients, this one with erlotinib plus tivantinib (ARQ 197) (MET-TKI) versus just erlotinib alone in previously-treated NSCLC patients showed that the median PFS was longer in the erlotinib plus tivantinib group than in the erlotinib alone group, particularly among patients with nd point . Based oMET gene status and clinical outcome for EGFR-TKI should be further evaluated using larger scale samples.Pre-gefitinib MET FISH status may be useful for predicting PFS and OS after Gefitinib treatment in lung adenocarcinoma with EGFR mutation and for selecting the patients who would benefit from EGFR-TKI and MET inhibitor therapy. Correlations between


We present a case of immunoglobulin G4 (IgG4)-related disease with pancreatic andextrapancreatic involvement, including the biliary and renal systems. Given theimportance of imaging methods for the diagnosis of IgG4-related disease and itsdifferentiation from pancreatic adenocarcinoma, we emphasize important abdominalcomputed tomography and magnetic resonance imaging findings related to thisrecently recognized systemic autoimmune disease. Although the full range ofvisceral involvement has yet to be fully described, involvement of the salivaryglands, periorbital tissues, meninges, lungs, aorta, pericardium, breasts, thyroid,and skin have been reported. Given that it most commonly affects the pancreas,IgG4-related disease commonly presents clinically as abdominal discomfort,obstructive jaundice, and weight loss, the differential diagnosis with pancreaticadenocarcinoma being important,4.Immunoglobulin G4 (IgG4)-related disease is a systemic autoimmune disease withpancreatic and extrapancreatic abdominal manifestations; it can affect the bileducts, kidneys, lymph nodes, prostate, and retroperitoneum, occurring primarily inthe 6th and 7th decades of lifeThe diagnosis of IgG4-related disease is based on clinical, radiological, andpathological findings. Given the importance of imaging methods for the diagnosis ofIgG4-related disease, we report a case of IgG4-related disease, highlighting themain computed tomography (CT) and magnetic resonance imaging (MRI) findings.A 49-year-old male patient presented with pruritus, abdominal discomfort, jaundice,choluria, hypocholic stools, and weight loss (of 13 kg in five months). Physicalexamination revealed jaundice and no fever, as well as a soft abdomen that waspainful on palpation of the right side. The patient had a history of smoking andalcoholism. Laboratory tests showed elevated liver enzymes and bilirubin levels(with an obstructive pattern), serum amylase and lipase levels being within thenormal range.After an ultrasound showing bile duct dilatation, an endoscopic retrogradecholangiopancreatography revealed common bile duct stenosis. An endoscopicpapillotomy was performed, and a biliary stent was placed. Brush biopsy of the bileduct was negative for neoplasia.Abdominal CT and MRI scans showed diffuse pancreatic enlargement with loss of thenormal pancreatic lobulation and roughness, a hypoenhancing peripancreatic halo inthe tail of the pancreas, and hypoenhancing renal nodules with partially ill-definedborders  and 2. MGiven the suspicion of IgG4-related disease, renal and pancreatic biopsies wereperformed. Examination of the biopsy samples showed dense, mixed lymphoplasmacyticinflammatory infiltrate with no evidence of epithelial neoplasia, most of the plasmacells in the infiltrate being IgG4-positive. Taken together, the imaging andhistopathological findings confirmed the diagnosis of IgG4-related disease; thepatient was started on corticosteroid therapy, which resulted in significantclinical and radiological improvement after approximately 16 weeks . At this. It is characterized by lesions showingfibrosis-associated inflammatory response and lymphoplasmacytic infiltration rich intissue IgG4-positive plasma cells, constituting a spectrum of fibroproliferativediseases including autoimmune pancreatitis, sclerosing cholangitis, sclerosingsialadenitis, sclerosing mesenteritis, inflammatory pseudo-tumor, andretroperitoneal fibrosis.IgG4-related disease is a recently recognized disease that was described as aspecific clinical and pathological entity in 2003.According to the international consensus diagnostic criteria for autoimmunepancreatitis, there are two specific subtypes of autoimmune pancreatitis (types 1and 2), which can be differentiated on the basis of five cardinal criteria: a)imaging changes in the pancreatic parenchyma and duct; b) serology (for IgG4 and IgGantinuclear antibodies); c) extra-pancreatic involvement; d) histology; e) responseto corticosteroid therapy-4, all of which were observed in the case presented here. Incases of focal or multifocal involvement, there are relatively well-defined lesionsassociated with ductal stenosis and upstream dilatation.The diagnosis of type 1 and 2 autoimmune pancreatitis can be definitive or probable,and, in many cases, the distinction between the two subtypes is impossible. Type 1autoimmune pancreatitis is also known as lymphoplasmacytic sclerosing pancreatitisand is the most common manifestation of IgG4-related disease, pancreatic involvementbeing classified as diffuse, focal, or multifocal. In the case presented here, aprobable diagnosis of type 1 autoimmune pancreatitis was established. Diffusepancreatic involvement is the most common type of pancreatic involvement andincludes diffuse pancreatic enlargement with loss of the normal pancreaticlobulation and roughness (a "sausage-like" pancreas), which is due to thelymphoplasmacytic infiltrate and obliterative phlebitis associated with IgG4-relateddisease. On MRI scans, the pancreas appears slightly hyperintense on T2-weightedimages and hypointense on T1-weighted images; a thin, hypointense halo is sometimesseen on T1- and T2-weighted images. In 16-80% of cases, there is delayed, mildhomogeneous enhancement after contrast administration, with a thin, hypoenhancingperipancreatic halo, indicating inflammatory and fibrotic changes in peripancreatictissues, as well as irregular narrowing of the main pancreatic duct-4; the intensity of contrastenhancement, which is lower in cases of adenocarcinoma than in cases ofpancreatitis; andapparent diffusion coefficient (ADC) values, which are lower in cases ofpancreatitis than in cases of adenocarcinoma, a cut-off ADC value of 1.075 ×10-3 mm 2/s having been suggested for differentiatingbetween the two entities. Casesof diffuse pancreatic involvement should be differentiated, first and foremost, fromacute pancreatitis; in cases of IgG4-related disease, there is a peripancreatichalo, little or no peripancreatic densification, and no fat necrosis. In most cases,however, a combination of clinical, laboratory, imaging, and histological criteriais required for such differentiation-4.Autoimmune pancreatitis can mimic pancreatic adenocarcinoma, especially when thedisease is limited to the head of the pancreas (as it is in approximately 80% ofcases). The two entities can be differentiated by determining the following: whetheror not the main duct penetrates the mass-it does in cases of pancreatitis, but itdoes not in cases of neoplasia-4. These findings are similar to those in patientswith primary sclerosing cholangitis (PSC), the differential diagnosis between thetwo entities posing a challenge. Patients with PSC are generally less symptomatic,whereas those with IgG4-related sclerosing cholangitis have disease that is moreacute and of a shorter duration. Patients with PSC have multifocal involvement ofshort segments including intrahepatic or extrahepatic bile ducts-normal segmentsalternating with slightly dilated segments to produce a "beaded" appearance-whereaspatients with IgG4-related sclerosing cholangitis have strictures that typicallyaffect a long segment and are continuous with prestenotic dilatation-4. Given this challenge, the American Association for the Studyof Liver Diseases recommends that serum IgG4 levels be measured in all patients withpossible PSC in order to exclude IgG4-related sclerosing cholangitis.With regard to extrapancreatic manifestations, the biliary tree is the most commonsite of involvement; bile duct wall thickening, stenosis, irregularity, and upstreamdilatation can occur, the intrapancreatic portion being the most commonly affectedsite,8. These imaging findings reflect histopathological featuresincluding lymphoplasmacytic infiltration of the renal interstitium, with anincreased number of IgG4-positive plasma cells, and fibrosis,8. The combined presence of pancreatic and extrapancreaticmanifestations further supports the diagnosis of IgG4-related disease, as in thepresent case.Renal involvement occurs in up to one third of patients and is usually multiple andbilateral, presenting as round or wedge-shaped cortical nodules, small peripheralcortical lesions, renal masses (pseudotumors), or renal pelvic involvement.Contrast-enhanced CT typically shows hypodense lesions showing mild contrastenhancement in later phases. On MRI scans, the lesions are isointense/hypointense onT1-weighted sequences and hypointense on T2-weighted sequences. Although the number of studies onthis topic is growing, the natural history and long-term prognosis of IgG4-relateddisease have yet to be well defined. Therefore, patient monitoring and a multisystemevaluation approach, including periodic imaging, are required.After clinical improvement with corticosteroid therapy, patients with IgG4-relateddisease require follow-up, given that there have been reports of an increasedrelative risk of cancer in the year following diagnosis-including stomach cancer(the most common type of cancer in such patients), lung cancer, prostate cancer,colon cancer, non-Hodgkin lymphoma, bile duct cancer, and thyroid cancerGiven that IgG4-related disease can mimic neoplastic lesions, there is a risk that amore aggressive approach might be taken if a diagnosis of IgG4-related disease isnot taken into account. The case reported here shows that radiologists should beaware of this possibility in order to establish the correct diagnosis and select themost appropriate therapy.


Large livestock farms might increase the infection risk for the nearby human population because of an increased risk for disease outbreaks and because antibiotic-resistant bacteria are more likely to be present. We hypothesized that populations residing in rural areas have more contact with cattle compared with populations in urban areas, and will use more antibiotics or more frequently require a new course of antibiotics.Using data from the prescription database IADB.nl, we compared antibiotic use by patients living in rural areas to the use by patients living in urban areas. We also followed cohorts of antibiotic users and determined the patients who required a second antibiotic within 14 days after beginning the first antibiotic.The yearly prevalence of antibiotic use was greater in rural areas compared with urban areas (2009: 23.6% versus 20.2% (p < 0.001), especially in the younger age groups. More adult patients residing in rural areas required a second course of antibiotic treatment within 14 days after starting the first treatment.Individuals use more antibiotics, and adults more frequently require a second antibiotic prescription within 14 days, in rural areas compared with urban areas. Although the differences were small and the risks for the general rural population were not high, this difference should be investigated further. Over the last 20 years, the number of farms have decreased while the numbers of animals have increased. This increase has been especially significant for animals , which are kept in large numbers in concentrated growout facilities . ConcentCoxiella burnettii) in goats in the southern Netherlands [Staphylococcus aureus (MRSA) [Escherichia coli strains from dairy farms have caused serious infections in humans in Belgium and the United States [In 2009, the nearby human population was affected by an outbreak of Q fever (s (MRSA) ,4 results (MRSA) . Highly d States ,7.In addition to these specific outbreaks, there may be general effects on the health of populations living in areas near livestock farms. There is little information about whether, compared with people in urban areas and others who are not typically in close contact with cattle, infections are more common, antibiotic use is greater, and antibiotics are more likely to be ineffective in populations that reside in rural areas.Using a pharmacy prescription database, we investigated the differences in antibiotic use between urban and rural areas. We used the need for a new antibiotic prescription within 14 days after starting a course of treatment as a signal for therapeutic failure.Information on drug use was obtained from the IADB.nl database, which contains pharmacy-dispensing data from community pharmacies in the Netherlands. Dutch patients usually register at a single community pharmacy, so a single pharmacy provided an almost complete listing of each subject’s prescribed drugs . The phaThe data in the IADB.nl database were strictly anonymous. No identification of individuals was possible. According to Dutch regulations, ethical approval was not required for the study .To perform this study, we required additional information on the environments in which the subjects resided. We used degree of urbanization (DU) information maintained by Statistics Netherlands  . In the Every prescription in the IADB.nl database was linked to the first four numbers of each subject’s postal code . We selected all Postal Code-4 areas in which ≥60% of the population was included in the IADB.nl database.The DU was linked to neighborhoods in the Statistics Netherlands database, not to postal code areas. There was only partial correspondence, and in most cases differences and overlaps were present. To resolve this problem, we determined the neighborhood with the lowest DU value  in each Postal Code-4 area. The Postal Code-4 area was then assigned this DU value. To maintain anonymity of the data, the postal codes were deleted from the data set after the DU values were assigned.To find an optimal effect, we selected the Postal Code-4 areas with a DU of 1 (n = 24) (‘urban’) and with a DU of 5 (n = 28) . The rural areas were identified as agricultural areas using the software application Google Earth . We selected all antibiotic drug prescriptions that were dispensed in these areas (ATC-code starting with J01) between 1998 and 2009.The number of prescriptions per person residing in both the rural and the urban populations was determined for each year (1998 to 2009). We specifically examined the prescriptions for 1999, 2004, and 2009, and calculated the prevalence of the population using at least one course of antibiotics per year, stratified by age and sex. For these 3 years, we also calculated the average number of prescriptions per antibiotic user. We also determined the proportions of the prescriptions of the different types of antibiotics for the rural and urban populations.Therapeutic failure was investigated by determining whether a second course of antibiotics was prescribed within 14 days of the start of a new course of antibiotics. A course of antibiotics was defined as new if the patient was not prescribed antibiotics during the preceding 30 days. To determine therapeutic failure, two cohorts of patients  who started an antibiotic course between 1 January 1999 and 31 December 2009 were selected. We compared the rural cohort with the urban cohort in an age-stratified analysis, and estimated the relative risks (RR) of needing a new prescription within 14 days after beginning the new treatment.U test was used to compare the numbers of prescriptions per person. Relative risks (RRs) with 95% confidence intervals (CI) were used to compare therapeutic failure. Microsoft® Office Excel 2010 , SPSS 16.0 for Windows , and R version 2.13.0  software were used for the data analysis. A p-value < 0.05 was considered to be statistically significant.The Chi-square test was used to compare percentages. The Mann-Whitney A total of 141,866 prescriptions for antibiotics for Postal Code-4 areas with a DU value of 5  were selected from the database. A total of 569,946 antibiotics prescriptions for Postal Code-4 areas with a DU value of 1 (‘urban’) were selected. The results for the 2009 data for population characteristics are presented in Table Table The results for the number of prescriptions per antibiotics user are presented in Table The cephalosporins and the fluoroquinolones are mostly distinguished as ‘reserved’ antibiotics to prevent antibiotic resistance. These two types of antibiotics were prescribed more often in urban areas Table . CompareThe estimated RRs for being prescribed a second course of antibiotics within 14 days of beginning a new antibiotic are presented in Table We found that the use of antibiotics per year was higher in rural areas compared with urban areas Figure . This diThe empirical probability of medication use, especially antibiotics, is also associated with socioeconomic status ,12. In mThe use of cephalosporins and fluoroquinolones was more frequent in urban areas, which was not the result we expected. The close proximity of hospitals and the availability of specialized care could have contributed to this result.No differences were found in children for the RR of needing another antibiotic, but the RR significantly exceeded 1 in the older age groups. Therapeutic failure of antibiotics seems to occur more often in older individuals living in rural areas, compared with those living in urban areas. The RR values were very close to 1, which implies that the measured effect was small. The effect might have been diluted by rural patients who did not have regular contact with cattle.No other studies have been published for populations from the Netherlands that compare the use of antibiotics in urban and rural areas. Because urban and rural areas are characteristically very dissimilar in different countries, the results of studies performed in other countries are difficult to compare with the results of our study.Two studies that investigated health symptoms associated with rural areas were performed in the Netherlands. Investigators that examined a population in the southern Netherlands compared the health of individuals residing near intensive livestock farms with that of individuals in other regions . They reTwo advantages of our study were that we used objective pharmacy data and large population sizes. One study limitation was that some specific population data was missing. The database consisted of pharmacy prescription data, but did not include information on indications. The study was also limited by its assumption that inhabitants of rural areas were more likely to reside in closer proximity to cattle. We assumed that because most farmers reside on their farms, the chance that a patient worked with animals was greater compared with urban residents.Compared with young people residing in urban areas, young people that resided in rural areas were more likely to use antibiotic drugs. Adults in rural areas more frequently required a second prescription of antibiotics within 14 days after beginning the first prescription. This result suggests that therapeutic failure due to antibiotic resistance may be a factor in this group. The differences between groups were quite small, probably because the effects were diluted. The risk for the general rural population is probably not significant. However, the factors that may have contributed to these results should be investigated in greater detail.The data in the IADB.nl database are strictly anonymous. No derivation to any recognizable person is possible. Ethical approval was not required, according to Dutch regulations.The authors declare that they have no competing interests.JJ performed the study and wrote the manuscript. JB, an IT-specialist and programmer, developed the databases and assisted in selection and analysis of the data. TV participated in the design of the study, and in the analysis and preparation of the manuscript. LJB supervised and provided advice during the performance of this study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/677/prepub


Antimicrobial resistance is a serious global threat that needs urgent attention. Antimicrobial stewardship (AS) programs were proven to be effective in reducing antibiotic days in adults. Pediatric-specific data about the effectiveness of implementation of such programs are limited especially in the Middle East.Our primary objective was to identify the top three antibiotics and measure their specific days of therapy (DOT) per 1000 pediatric intensive care unit (PICU) days per quarter before and after the AS intervention.We conducted a pilot prospective quality-improvement interventional study in a 20-bed PICU in a specialized tertiary care children’s hospital in Riyadh, Saudi Arabia from April to December 2014. Data about antimicrobial indication and utilization were gathered from study-designed forms. An AS member from pediatric infectious disease team rounded three times per week on all patients in PICU and provided antibiotic-related recommendations based on available bedside information and ensured adherence to antimicrobial restriction policy. Intervention also included educational sessions to physicians and nurses about proper use of antibiotics.During the study period, 648 children out of 898 PICU admissions (72.2%) were utilizing antimicrobials. Vancomycin, Pipracillin/Tazobactam and Meropenum represented approximately 60% of the 36 antimicrobials used. Per quarter, antibiotic-specific DOT dropped from 348.5 to 320.5  for Vancomycin, 356.2 to 294.7  for Pipracillin/Tazobactam, 162.0 to 111.1  for Meropenum.Education and bedside real-time AS recommendations significantly dropped the use of Meropenum, Pipracillin/Tazobactam and Vancomycin. Moreover, AS program proved to be effective and promising in reducing antibiotic days in PICU.None declared.