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The German version of the Sociocultural Attitudes toward Appearance Questionnaire (SATAQ-G, Knauss et al., 2009) is an instrument used to assess the influence of sociocultural body ideals on body image. For this study, the only subscale administered was the boys’ version of the internalization of media body ideals subscale (α = 0.84). Convergent validity was reported to be acceptable (Knauss et al., 2009). The internalization subscale consists of six items rated on a five-point Likert scale from 1 (strongly disagree) to 5 (strongly agree), with higher mean scores indicating a higher internalization of a muscular, lean male body ideal.

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To assess participants’ history of stressful peer experiences (e.g., teasing), the German questionnaire for experience of peer victimization in childhood and adolescence (FBS, Sansen et al., 2013) was used. The FBS has been shown to have good validity and test-retest reliability (r = 0.83; Sansen et al., 2013). This 22-item retrospective questionnaire is based on a binary response format (experienced/ not experienced). Construct validity was confirmed via correlations between the FBS and psychological distress and social anxiety measures (Sansen et al., 2013). Calculating the overall sum value, higher scores indicate higher levels of bullying experience.

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The desire to obtain a bigger and more muscular physique was assessed with the German translation of the Drive for Muscularity Scale (DMS, Waldorf et al., 2014). The self-rating questionnaire contains 15 items. It can be divided into two subscales, muscularity-related attitudes (DMS attitudes, eight items) and muscularity-related behavior (DMS behavior, seven items). The items were rated on a six-point Likert scale from 1 (always) to 6 (never) and were reverse-coded with higher mean scores representing greater drive for muscularity. For the original version, with no distinction between the subscales, internal consistencies were reported to be good for both men (α = 0.90) and women (α = 0.83; McCreary and Sasse, 2000). In the German version internal consistency in different samples was good (global scale α = 0.89 to 0.90, muscularity-related attitudes α = 0.88 to 0.90, muscularity-related behavior α = 0.79–80; Waldorf et al., 2014). Retest-reliability was also good (global scale r = 0.95, muscularity-related attitudes r = 0.92, muscularity-related behavior r = 0.96) and validity was confirmed using a correlation (r = 0.81) with the Male Body Attitudes Scale (Waldorf et al., 2014). In the present sample, Cronbach’s alpha for the global scale was 0.89, for the muscularity-related attitudes 0.90, and for the muscularity-related behavior 0.75.

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Participants were recruited through advertisement in online panels for the German-speaking weight-training and bodybuilding community. German, Austrian, and Swiss panels were provided with a web link to the survey, which took approximately 30 min to complete. Before starting the online survey, participants were informed about the study and its purpose. All participants took part on a voluntary and anonymous basis and were treated in accordance with the declaration of Helsinki. After completion of the survey, which was provided only in German language, participants were given the possibility to take part in a lottery with the chance of winning one of two 50 € vouchers for dietary supplements as an incentive.

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Prior to analyses, scores on the predictor variables and the DMS were examined for outliers. We z-transformed and checked all outcome variables for values out of an absolute variation of three standard deviations. No unacceptable values were being found1. All assumptions for regression analysis have been met, specifically no multicollinearity, the normality of the dependent variables, no autocorrelations, and homoscedasticity of the residuals.

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Using a hierarchical forced entry method, three multiple linear regression analyses were conducted. All variables were investigated regarding their relation with (1) drive for muscularity, in addition to each of the (2) muscularity-related attitudes, and (3) muscularity-related behavior subscales. Since there is only marginal evidence for the association with biological factors, such as age or FFMI (Cafri et al., 2005), these variables were added separately. Thus, all three regression analysis were conducted with two steps each, (1) one for the biological factors and (2) one for the psychological and sociocultural factors.

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As shown in Table 1, respondents stated exercising a mean of 3.96 times per week (SD = 7.37) for 79.87 min per session (SD = 23.93). The mean of years of exercise was 5.05 (SD = 5.45). Average BMI indicated slight overweight status (M = 25.57, SD = 2.93); however, body fat percentage (M = 13.36, SD = 3.49) was in the lower healthy range for men between 20 and 39 years of age (Gallagher et al., 2000). Compared to a study of the general population of Austria (M = 25.9), this sample had a comparable BMI value (M = 25.57), but a much lower body fat percentage (M = 13.36 vs. M = 22.3; Elmadfa et al., 2012). This shows that the BMI is only partly useful within a population of highly muscular individuals, and other body composition measures are needed.

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The mean value for the FFMI was 22.09 (SD = 2.54). Based on the classification of Pope et al. (2001), 6.4% of the men described their musculature as below average, 30.6% as average, and 62.8% as above average. 8.9% of the sample disclosed former or current use of illegal, physically enhancing supplements (e.g., AAS) to become more muscular. The majority pursued the goal of gaining more muscle mass (84.7%), improving physical appearance (79.4%), increasing well-being (74.6%), promoting health (72.6%), and increasing strength (69.8%). Further aims included the reduction of body fat (43.5%), performance improvement in other sport activities (20.6%), participation in bodybuilding competitions (15.7%), and weight loss (8.1%). In addition, 88.3% of all participants indicated dieting in order to achieve lean muscularity.

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As presented in Table 2, along with the semi-partial correlations (the factors unique contribution to the outcome), age was the strongest negative predictor (i.e., the predictor greatest in magnitude if the regression coefficients) for aspects of drive for muscularity, while FFMI revealed the second-strongest positive connection with drive for muscularity-related behavior. On the other hand, FFMI did not predict muscularity-related attitudes. Both factors accounted for 9–14% of variance in drive for muscularity and its related attitudes and behaviors.

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Regarding the psychological factors both body-related self-esteem facets, physical attractiveness and fitness significantly predicted overall drive for muscularity. They also predicted muscularity-related attitudes and muscularity-related behavior. In all cases they were negative predictors, in that lower self-esteem facets predicted higher values of drive for muscularity and its subscales.

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Global and performance-related self-esteem failed to predict drive for muscularity, although on a correlational level, significant relations have been found (global self-esteem: r = -0.26 to -0.52, p < 0.001; performance related self-esteem: r = -0.16 to -0.31, p < 0.01).

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Dissatisfaction with muscularity significantly predicted muscularity-related attitudes but not muscularity-related behavior in that higher levels of dissatisfaction with muscularity predicted higher values of muscularity-related attitudes. Dissatisfaction with body fat on the other hand, was no significant predictor for drive for muscularity, although it was significantly related to it on a correlational level (r = -0.18 to -0.22, p < 0.01).

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The strongest positive predictor for overall drive for muscularity, muscularity-related attitudes, and behavior was internalization of the media body ideal. Stressful social experiences were not significantly predicting drive for muscularity, nor one of its subscales. It showed only small significant correlations to drive for muscularity (r = 0.15) and its attitudinal aspects (r = 0.18).

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In conclusion, psychological and sociocultural variables accounted for 60% of variance in drive for muscularity, 64% of the variance in attitudinal, and 41% of the variance in behavioral aspects of drive for muscularity. Therefore, dissatisfaction with muscularity, body-related self-esteem and internalization of media body ideals enhanced the model significantly, leading to a 31% change in R2 in behavior, 50% in attitudes, and 48% in total drive for muscularity.

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Using a biopsychosocial model as a framework, the aim of this study was to investigate the associations of biological, psychological, and sociocultural variables with drive for muscularity in weight-training men. As predicted, these variables explained a significant amount of the variance in drive for muscularity and its subscales. In support of the hypotheses, internalization of media body ideals, dissatisfaction with muscularity, and aspects of self-esteem, which include physical attractiveness and fitness, as well as age and FFMI, each demonstrated significant predictors for weight-training men’s drive for muscularity. On the contrary, dissatisfaction with body fat, global self-esteem, performance-related self-esteem, and stressful peer experience were not significantly associated with drive for muscularity.

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Consistent with previous research on school and college samples (Daniel and Bridges, 2010; Stratton et al., 2015; Edwards et al., 2016), internalization of media body ideals was the strongest predictor of drive for muscularity and its related attitudes and behavior in weight-training men. Mass media play a key role in distributing an unrealistic muscular and lean male body image, which becomes the body ideal for many boys and men trying to achieve this goal by any means (Smolak et al., 2005; Cafri et al., 2006; Parent and Moradi, 2011; Readdy et al., 2011). To our knowledge, there are no studies explicitly investigating the impact on internalization of media body ideals in weight-training samples so far, but it can be assumed, that this sample shows higher degrees of internalization, since they might also be closer to this ideal than the usual samples of school boys and college or community men. Although this is only of descriptive value, we have found one study (Parent and Moradi, 2011) also using the internalization subscale of the SATAQ, showing a quite lower mean (M = 1.75, SD = 0.71) in a sample of college men than our sample (M = 2.88, SD = 0.93). Since increased levels of internalization were associated with increased drive for muscularity, it appears useful to develop prevention approaches for the weight-training community helping to reveal the potential harm that comes with the belief in, and massive consumption of, unrealistic body ideals.

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As a scarcely investigated factor, age showed to be a strong negative predictor for drive for muscularity. Increased age was related to lower global, attitudinal, and behavioral facets of drive for muscularity. It is possible that, over time, other aspects of life, such as career, financial resources, or family become more important than physical appearance, resulting in a reduction of drive for muscularity. Alternatively, the desired level of muscularity might be attained and the initial need be satisfied. The relation of age and drive for muscularity could be moderated by different degrees of internalization of media body ideals. Current data support Grieve’s (2007) notion that the muscular ideal appears to be very salient for adolescents and college men, but might be less important to older samples. This is consistent with Stratton et al. (2015), who found men older than college age to be less dissatisfied by bodily comparison. Self-esteem and body dissatisfaction could also be interacting with age, since it has been shown, that in adult life with higher age, both, self-esteem (Robins et al., 2002) and body satisfaction (Tiggemann and McCourt, 2013) are rising. Future research, especially longitudinal studies, are required to further examine the relationship between age and drive for muscularity, as well as developmental and hormonal influences in younger age.

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In line with other findings (McCreary and Sasse, 2000; Chittester and Hausenblas, 2009) we found relations between certain facets of low self-esteem and drive for muscularity. While fitness-related self-esteem represents to what extent individuals are content with their athletic and coordination skills, physical attractiveness-related self-esteem symbolizes the satisfaction with one’s appearance and a person’s confidence to be attractive (Schütz and Sellin, 2006). Both showed a negative relation with drive for muscularity. Fitness-related self-esteem was related to all facets of drive for muscularity. Physical attractiveness related self-esteem was associated only with global and attitudinal, but not behavioral aspects. This could result from peoples’ assumptions that physical attractiveness is often associated with facial attractiveness (Currie and Little, 2009), so that there might be no expectation that exercising could have an influence on physical attractiveness-related self-esteem. Global self-esteem and performance-related self-esteem, although significantly correlated to drive for muscularity, did not reach statistical significance in the regression analysis, pointing to the vital influence of body-related aspects of self-esteem in opposition to more general self-esteem variables. Although, it has been found that weight-training and bodybuilding samples report higher self-esteem than controls (Pickett et al., 2005), body related self-esteem seems to be important for the desire to become more and more muscular, which is of significance especially in populations with higher risk for MD.

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To our knowledge, this is one of the few existing examinations of dissatisfaction with muscularity as well as with body fat in relation to drive for muscularity in weight-training men. According to the literature (Blouin and Goldfield, 1995; Hildebrandt et al., 2004; Olivardia et al., 2004), it was assumed that both aspects of body dissatisfaction would be associated with drive for muscularity. Even though stronger dissatisfaction with muscularity was linked to global and cognitive aspects of drive for muscularity, it showed no relation with behavioral facets. This is in line with findings of Stratton et al. (2015), but contradicting those of Karazsia and Crowther (2010) and Tylka (2011), who found that dissatisfaction with muscularity significantly predicted muscularity-related behavior in college and community men. A reason for this could be that cognitive mechanisms and actual behavior are very distinct features. Just as not all women who are dissatisfied with their weight start dieting, not all men who are dissatisfied with their muscularity start increasing their muscularity-related behavior, especially when this behavior is already above average. Future research should investigate these two facets separately, especially regarding extreme forms of drive for muscularity.

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Dissatisfaction with body fat failed to predict drive for muscularity. According to Tylka (2011), dissatisfaction with musculature is related to muscularity-related behavior, while dissatisfaction with body fat is associated with disordered eating behavior. Since body dissatisfaction in general and drive for muscularity have been found to be distinct concepts (Bergeron and Tylka, 2007), it seems plausible that drive for muscularity can be understood as a strategy to reduce dissatisfaction with muscularity. Thus, dissatisfaction with body fat, related to different behaviors of coping (e.g., dieting, cardio workout) is unrelated to drive for muscularity.

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Even though some researchers have presented the advantages of the FFMI over the BMI (Cafri et al., 2005), to our knowledge, only two studies (using college samples) have systematically analyzed the FFMI in relation to drive for muscularity (McCreary et al., 2006; Chittester and Hausenblas, 2009). In contrast to Chittester and Hausenblas (2009) who did not detect any relations, we found an association of FFMI with muscularity-related behavior, but not with attitudes. This is partly in line with findings of McCreary et al. (2006). It appears that higher muscle mass is related to more muscularity-related behaviors. Thus, current results suggest that muscle growth relates to the behavioral, but not to attitudinal features of drive for muscularity.

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Future research needs to focus on a standardized operationalization and interpretation of the FFMI, since no clear instructions have been published and existing interpretations of the FFMI are inconsistent. Clearly, replication with the use of skin calipers or hydrostatic weighing (Cafri et al., 2005) instead of self-reported body composition measures is recommended.

other

Contrary to the hypotheses, stressful social experiences in childhood and adolescence were not associated with drive for muscularity, although a significant small positive correlation was found. These results are inconsistent with other findings (Nowell and Ricciardelli, 2008; Schaefer and Salafia, 2014). Thus, the assumption that men taking up intense exercise do so in order to make up for earlier stressful experience (Wolke and Sapouna, 2008) and use weight training as a coping strategy (Boyda and Shevlin, 2011) can be questioned.

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Yet, it is possible that stressful social experience serve as a moderator or mediator. Lamanna et al. (2010) argued that regular critique and emotional victimization through parents leads to higher degrees of body dissatisfaction, which again, is associated with drive for muscularity. Also, exercise as a coping strategy for more recent stressful experiences might be of more relevance than for those from the past. Therefore, including stressful social experience through peers, parents, and partners, especially related to body shape, might be useful in future research.

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A number of strengths can be named for the current study. Since the majority of research in this field uses school or college samples, the recruitment of a sufficiently large sample of weight-training men can be considered to be more meaningful for those persons at high risk for body image problems. Also, the investigation of biological, psychological and sociocultural factors simultaneously, using a biopsychosocial framework, allowing assumptions on different degrees of importance for drive for muscularity, can be useful. Further, investigating biological factors, such as the FFMI and age separately, provides important directions for future research. Especially, the effect of age, along with hormonal influences should be investigated on a longitudinal level, since changes in its relation to drive for muscularity can be assumed as being non-linear.

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The current study also has a number of limitations. As described regarding the influence of age, a longitudinal approach could lead to more valid findings. A cross-sectional design, as used in this study, limits inference with regard to the temporal order in which the factors may operate. Also, online studies rely on self-reported measures, which are especially prone to biases in regard to body measures. Although, bodybuilders and weight-trainers can be assumed to have more precise knowledge about their bodies than the average person, on-site measuring would be preferable and should be used in future studies.

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Another limitation concerns the homogeneity of the sample. First, since we used a weight-training sample, no generalization on the general population can be drawn. Since it is usually weight-training and bodybuilding men who are at risk of pathological outcomes of extreme drive for muscularity, the results might still be valuable for prevention and treatment in this special population. Second, results are only generalizable for men with relatively low body fat percentages, since the FFMI was only interpretable when body fat percentage was below 20%. Third, although our sample is, on average, 5 years older than the usual college sample, no conclusions can be drawn for weight-training men in older age categories. Moreover, the vast majority of the current sample is from Western, industrialized, German-speaking countries and generalization is only viable for these populations. Furthermore, there was a very low percentage of homosexual or bisexual men in this sample (3.2%). Since homosexuality is discussed as a risk factor for eating disorders in men (Russell and Keel, 2002; Freeman, 2005), it would be interesting to investigate its relation to drive for muscularity further.

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This study aimed at further investigating factors associated with drive for muscularity in weight-training men, using a biopsychosocial model as a frame. Our current findings confirmed most of the hypothesized factors as being associated with global drive for muscularity and muscularity-related attitudes and behaviors. Internalization of media body ideals presented the highest predictive value for drive for muscularity. This indicates that the drive for muscularity, similar to the drive for thinness in women, seems to be a problem of societies in which highly unrealistic body ideals are advertised and idealized. As a consequence, education on, and advertisement of, body ideals based on health and well-being, instead of thinness or leanness and muscularity, should be pursued in order to promote health instead of appearance.

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The study was conducted in accordance with the principles of the Declaration of Helsinki and with institutional guidelines of the School of Psychology, University of Vienna. Furthermore, it followed the Guidelines of the ethical conduct proposed by the American Psychological Association. According to the institutional guidelines of the University of Vienna, Austria (http://satzung.univie.ac.at/ethikkommission-der-universitaet-wien/), approval by an ethics committee was not necessary because the study did not affect the physical or psychological integrity, the right for privacy, or other personal rights or interests (see § 2(1)). All participants gave informed consent on the first page of the online survey (prior to the actual start of the study) after having received a written description of the study. Data collection was anonymous and no harmful procedures were used. Furthermore, participation was voluntarily and no remuneration for participation was given.

other

CS and LR designed and conducted the study in consultation with KH-F. CS and LR analyzed the data with assistance and contributions from MV and KH-F. CS drafted the manuscript with contributions from LR, MV, and KH-F. All authors read and approved the final manuscript.

other

Recently, size switchable materials have attracted much attention, as size is the fundamental and crucial factor that determined the properties of materials, for example as the noble metal nanoparticles1, polymeric microparticles2 or colloidal superparticles3. The stimuli-responsive molecules might be very useful to build up the size switchable systems45. Azobenzene is one of the most attractive stimuli-responsive molecules67, because of its reversible isomerization between trans and cis conformation upon external stimuli, such as mechanical stress8, electrostatic stimulation910, as well as light irradiation111213. So the supramolecular assemblies involved azobenzene have various applications in intelligent membranes1415, optical memories161718 and biological systems192021 and so on. A large number of photo-responsive systems based on the azobenzene and macrocycles have been reported2223242526, such as, a photo regulable ion-extraction and ion-transport based on azobenzene-crown ethers27, a photo-responsive artificial muscle based on host-guest complex of α-cyclodextrin and azobenzene2829, and a dual stimuli-responsive self-assembly supramolecular nanoparticles based on the ternary host-guest complexation between cucurbituril, a methyl viologen polymer, and mono- and multivalent azobenzene functionalized molecules30.

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As a new class of macromolecules, pillar[n]arenes (5–15) have been extensively studied from their synthesis, functionalization and supramolecular chemistry since 2008313233343536. The first photo-responsive host-guest complexation based on pillararene and azobenzene-guest was reported by Huang in 201237. Thereafter, the author constructed a dual-responsive supra-amphiphilic polypseudo-rotaxane based on pillararene and azobenzene derivative in aqueous solution38. Meanwhile, Ogoshi also reported a photoreversible switching of the lower critical solution temperature (LCST) in host-guest system of pillararene and azobenzene derivative39. Up to now, most reported photo-responsive systems based on azobenzene and pillar[n]arene are mainly focus on pillararene404142. Nevertheless, the diameter of the internal cavity of pillararene is ~4.7 Å, which is equal to α-cyclodextrin (~4.7 Å)43. In accordance with common sense, it is reasonable that the pillararene and azobenezene derivative could form host-guest inclusion complexation. Ogoshi has studied the threading process of pillararene onto a viologen derivative containing an azobenzene end-group44. But there has been no direct proof to reveal the host-guest complexation between anionic pillararene and photo-responsive azobenzene derivatives. Considering the easier access to pillararene compared with pillararene and the aforementioned reasons, it is necessary to investigate the host-guest complexation and assembly behavior based on pillararene and azobenzene derivatives. In addition, to the best of our knowledge, size switchable supramolecular nanoparticles formed by host-guest interactions between anion pillararene and azobenzene derivatives have been underexplored.

review

Herein, we present a feasible strategy for fabricating photo/thermal-responsive size switchable supramolecular nanoparticles assembly based on the unprecedented host-guest inclusion complex between anionic pillararene 2C-WP5A and azobenzene derivation G in aqueous solution (Fig. 1). The design proposed here combines the following advantages: (1) the percarboxylatopillararenes is the most used anionic pillararene, which can be obtained easily and efficiently; (2) differing from the α-cyclodextrin, either trans- and cis- azobenzene derivatives G could be included in 2C-WP5A with similar affinity, which could result in the only reversible change of the nanoparticle’s size without disassembling; (3) the size change by photo/thermal-stimulation would result in the reversible switching the transparency of solution from turbid to clear.

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The host-guest complexation of 2C-WP5A and trans-G was first investigated by 1H NMR experiment in D2O at 25 °C (Fig. 2b). Signals for the protons on trans-G were significantly shifted upfield in the presence of 1 equiv. 2C-WP5A. This result suggested that the formation of the threaded structure 2C-WP5Atrans-G, in which these protons of the guest molecule were shielded by the aromatic rings in 2C-WP5A. The Job’s plot was employed to find the binding stoichiometry of 2C-WP5Atrans-G, the top point of the curve was appeared at the molar fraction Xguest = 0.5 (see Supplementary Fig. S12), indicating the 1:1 binding stoichiometry between 2C-WP5A and G.

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Interestingly, comparing to the chemical shift of the protons on the pyridinium unit, the signals of the protons on the azobenzene unit changed more obviously. The complexation-induced chemical shifts (Δδ) followed the order (see Supplementary Fig. S13): Hh > Hg > Hi > Hd > He = Hc > Hf > Hb > Ha. These shifts of methyl and both aromatic rings of the azobenzene unit were shielded by the pillararene, and the ring A is more shielded than others, which indicated that the aromatic rings of 2C-WP5A were located at the azobenzene unit of trans-G especially at ring A, instead of pyridinium unit. Although we failed to get the single crystal of the 2C-WP5Atrans-G, the 2D ROESY NMR experiment (see Supplementary Fig. S14) also supported our speculation of the binding mode. A 2D ROESY spectrum of an equimolar (5 mM) mixture of trans-G and 2C-WP5A in D2O was obtained to confirm the peak assignments. NOE correlation signals were observed between protons Hg, He and Hf on the azobenzene unit of trans-G and side chain protons H3 and phenyl moieties protons H2 on 2C-WP5A. The results showed that azobenzene unit was indeed threaded in the cavity of anionic 2C-WP5A, which was consistent with the above-mentioned 1H NMR results. To further confirm this result, we synthesized another guest molecule G′ with 1,4-diazabicyclo[2.2.2]octane group. The additional experiment results (see Supplementary Figs S15 and S16) also supported our conclusion that the azobenzene unit was positioned inside the cavity of 2C-WP5A. Recently, an inclusion complex of azo dye and cationic pillararenes was reported by Stoikov, which is also confirmed that azobenzene unit threaded in the cavity of pillararene45.

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To further investigation of the photo-responsive properties of the host-guest complex and supramolecular assembly of 2C-WP5A and G, we firstly examined the photoisomerization behavior of G by UV/vis (see Supplementary Fig. S17) and 1H NMR (Fig. 2d). The UV/vis spectrum of trans-G has a strong absorption maximum at 351 nm, which corresponds to the π-π* transition of the trans form. When the solution of trans-G was irradiated at 365 nm for 2 min, the main absorption band bleached greatly. Meanwhile, slightly increase in a new band at 432 nm, which is ascribed to the n-π* transition of the cis form of the azobenzene. The changes in the UV/vis spectrum indicated that the photoisomerization of G from the trans to cis conformation. Moreover, the trans/cis photoisomerization of G can be quantitatively calculated by integration of the signals in the 1H NMR spectrum. After irradiation with UV light at 365 nm for 10 min to ensure the fully transformed, a series of new signals appeared which is corresponding to the cis form, and the ratio of the trans:cis reached up to 10:90 (Fig. 2d). It is remarkable that G has not only photo-responsive but also thermal-responsive performances46. The continuous heating of the irradiated solution at 80 °C for one hour resulted in the regeneration of original protons signals of G, suggesting the recovery of trans form of G.

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After irradiation the solution of 1:1 2C-WP5Atrans-G with UV light at 365 nm for 10 min, some new signals appeared as shown in Fig. 2c. In comparison to the signals of individual G at the same experimental condition (Fig. 2d), we observed that the chemical shift of proton Hi* shifted upfield from 3.75 ppm to 1.71 ppm, and the peaks of the protons related to the azobenzene altered slightly (he*, hf*, hg* and hh* Δδ = 0.15, 0.06, −0.13 and −0.92 ppm, respectively). Those changes indicated that the ring B was deshielded by aromatic ring of pillararene, and the methyl group was more shielded than others, which implied the most of azobenzene unit was threaded out of pillararene for 2C-WP5Acis-G. The complexion of 2C-WP5AG exhibited photo-responsive and thermal-responsive as well, the reversible switchable process of the inclusion complex between 2C-WP5A and G could be modulated upon irradiation with UV at 365 nm and heating at 80 °C for one hour (Fig. 2e).

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To further understand the interaction between 2C-WP5A and trans/cis- G, we used isothermal titration calorimetry (ITC) to explore the thermodynamics of the host-guest inclusion complex (Fig. 3). The Ka values and thermodynamic parameters are listed in Table 1, and we are surprised to find that there was no obvious difference between the Ka-cis and the Ka-trans. The hydrophobic interaction is characterized by positive value of ΔH454748, which suggests that the hydrophobic interaction is the main force in the complexation. Meanwhile, 2C-WP5A and G immobilized their conformation during the complexation, resulted in a great negative entropy change which offset the positive entropy change aroused by solvent liberation49. This great unfavorable entropy make us easily understand why pillararene cannot bind with azobenzene in organic solution although its size is similar to α-cyclodextrin.

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Recently, we have developed a novel strategy for the construction of supramolecular assemblies by means of host induced aggregating (HIA: a small amount of macrocyclic host could promote the aggregation of guest molecules by lowering the critical aggregating concentration (CAC) and even regulating the morphology of the aggregates)5051525354555657. So after successful establishing the recognition motif between 2C-WP5A and G in aqueous solution, we were trying to explore the constructing supramolecular aggregates based on 2C-WP5A and trans-G. G could be induced by 2C-WP5A to form large assembly via HIA by monitoring the dependence of the optical transmittance at 600 nm on the concentration of G (There is no absorbance of G at 600 nm Fig. 4a). It is noteworthy that in the absence of 2C-WP5A, the optical transmittance of trans-G at 600 nm showed no appreciable change as the concentration increased form 0.8 mM to 2.0 mM (see Supplementary Fig. S18). It is requisite to find the best molar ratio between 2C-WP5A and G for fabricating the supramolecular assembly. As shown in Fig. 4b, the optical transmittance at 600 nm of the trans-G solution first decreased sharply upon the gradually added 2C-WP5A until reaching the minimum and then gradually increased as the amount of 2C-WP5A continued to increase. By monitoring the change of the transmittance at the wavelength of 600 nm in the process, the best molar ratio of trans-G and 2C-WP5A can be determined at 1:0.06. Redundant amount of 2C-WP5A leads to the formation of a simple inclusion complex and disaggregation of the aggregates, accompanied with the rising of the optical transmittance. This phenomenon highlighted the important role of HIA in the formation of the supramolecular assembly. In fact, only a small amount of macrocyclic host (6%) could promote the aggregation of guest molecules. In order to prove the prominent effect of the macrocyclic structure of host, a control experiment showed that no assembly appeared when replacing 2C-WP5A with its fragment benzene-1,4-dioxyacetic acid ammonium salt (DAAS) under comparable conditions (see Supplementary Fig. S19), indicating that the cyclic structure of 2C-WP5A is vital in inducing the supramolecular assembly. Furthermore, in order to make comparison with HIA, we fixed the concentration of 2C-WP5A at 1.6 mM, gradually added trans-G and controlled the experiment procedure same to the HIA. As shown in Fig. 4c,d, the transmittance the 2C-WP5A is not changed upon the addition of G from 0 to 1.0 eq. So for induced aggregating, the poly-charged additive is necessary such as the macrocyclic host molecules. Overall, there are three main points in HIA: (1) strong binding affinity between host and guest; (2) charge interaction between host and guest; (3) the macrocyclic structure of host molecule.

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Dynamic light scattering (DLS) and transmission electron microscopy (TEM) are used to characterize the size and morphology of 2C-WP5A+G assembly. As shown in Fig. 5a, DLS data showed that 2C-WP5A+trans-G formed well-defined aggregates with a narrow size distribution, giving an average diameter about 848 nm at a scattering angle of 90°. Meanwhile, the TEM image (Fig. 5c) also certificated the formation of spherical nanoparticles with diameter about 800 nm consistent with the DLS data. Furthermore, after irradiation the solution with UV light at 365 nm for 10 min, the solution became clear and the size of the aggregates formed by 2C-WP5A+cis-G changed into smaller nanoparticles with diameter about 253 nm (Fig. 5b,d). Afterwards, upon heating the solution at 80 °C for one hour, the size of the aggregates returned to the original status. So it is incontrovertible that the isomerization of G by photo/thermal stimulation has significant influence on the size and morphology of the supramolecular nanoparticles assembly.

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Zeta potential measurement was further performed to identify the supramolecular assembly surface charged distribution (see Supplementary Figs S20 and S21), both of the 2C-WP5A+trans/cis G assembly giving an average negative zeta potential to maintain the stability of the assembly. Based on the above experimental results and analysis, we postulate that the formation of the supramolecular assembly occurred in two steps. Firstly, the 2C-WP5A and G instantaneously formed a host-guest complex. Subsequently, excess guest molecules arranged around the host-guest complex due to electrostatic effect with redundant carboxylate anion of 2C-WP5A. According to the experimental results of the zeta potential, the 2C-WP5A was on the surfaces of the nanoparticles. Based on the above description, the poly-anionic structure of 2C-WP5A is very important in the formation of the supramolecular nanoparticles assembly.

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It is also required to make a thorough inquiry about the repetitiveness of the photo/thermal driven size switching of the supramolecular assembly. Size switching process was determined by monitoring the optical transmittance of 2C-WP5A+G solution at 600 nm (Fig. 6) and DLS experiment (see Supplementary Fig. S22). The presence of the large aggregates of the solution was evident from the decrease in transmittance, which was corresponding to the formation of spherical nanoparticles with diameter of 800 nm from DLS and TEM results. After irradiation with UV light at 365 nm for 10 min, the solution turned clear and transparent with nearly 100% transmittance. By continuous heating at 80 °C for one hour, the solution reverted to the initial turbidity state. Significantly, the reversible transition between turbidity and clarity could repeat several times, indicating that the good photo/thermal responsiveness of the supramolecular assembly.

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In conclusion, a novel recognition motif between anionic pillararene (2C-WP5A) and azobenzene derivative G has been developed, and both of the trans- and cis- G have the similar affinity with 2C-WP5A. The complexation process was well studied and confirmed by 1H NMR, 2D ROESY, and ITC experiment. The recognition motif would greatly expand the application in the area of construction of stimuli-responsive supramolecular assembly based on anionic pillararene. Furthermore, we utilized this novel recognition motif to construct supramolecular assembly nanoparticles which could reversibly switch the size from ~800 nm to ~250 nm by photo/thermal stimulation, presenting the turbid-to-clear switch of the solution state with excellent reversibility. To the best of our knowledge, this is the first example of supramolecular aggregate based anionic pillararene and azobenzene derivative. The high affinity of anionic pillararene with azobenzene derivative is expected to have great potential applications in further fabrication of more sophisticated stimuli-responsive supramolecular system.

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2C-WP5A was prepared according to the published procedures58. All reagents were commercially available and used without further purification, unless otherwise noted. Solvent were dried according to procedures described in the literature. Column chromatography was performed on silica gel (200–300 mesh).

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1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE AV400 (400 and 100 MHz). Signal positions were reported in part per million (ppm) relative to the residual solvent peaks or to the peak of Si(CH3)4 used as an internal standard with the abbreviations s, d, t, q, and m, denoting singlet, doublet, triplet, quartlet and multiplet, respectively. The residual 1H peak of deuterated solvent appeared at 4.79 ppm in D2O, at 7.26 ppm in CDCl3. All coupling constants J are quoted in Hz.

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The sample for TEM measurement was prepared by dropping the solution onto a copper grid. The grid was then air-dried. The samples were examined by a high-resolution TEM (Tecnai G2 F20 microscope, FEI) equipped with a CCD camera (Orius 832, Gatan) operating at an accelerating voltage of 200 kV.

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The ITC experiment was carried out at 25 °C in aqueous solution. In a typical experiment, the solution of 2C-WP5A in a 0.250 mL syringe was sequentially injected with stirring at 300 rpm into a solution of G in the sample cell (1.4227 mL). A control experiment to determine the heat of dilution was carried out by performing the same number of injections with the same concentration of host compound into ultrapure water. The dilution enthalpies determined in control experiments were subtracted from the enthalpies measured in the titration experiments to obtain the net reaction heat. All thermodynamic parameters reported in this work were obtained by using the “one set of binding sites” model. Two titration experiments were independently performed to give the averaged values with standard error (see Supplementary Fig. S23).

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Epidemics of meningococcal meningitis have been described in Africa since 1840 , and large-scale, cyclic epidemics have been described throughout what is called the African meningitis belt during the last century [2, 3]. Historically, the largest epidemics of meningococcal meningitis in the meningitis belt were caused by Neisseria meningitidis serogroup A (NmA). The introduction of a highly effective conjugate vaccine against NmA (MenAfriVac) in 2010 has had a major impact on the incidence of all-cause meningitis in the African meningitis belt , dramatic effects on the incidence of NmA , and has virtually eliminated its nasopharyngeal carriage .

review

In the relative calm after the introduction of MenAfriVac, small-scale epidemics caused by a novel strain of N. meningitidis serogroup C (NmC) were seen in northwest Nigeria in 2013 and 2014 . These two outbreaks were followed by a much larger epidemic in 2015 in both Nigeria (>6000 cases reported) and Niger (>9000 cases reported) .

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Two main strategies exist for meningitis prevention: mass vaccination (either in a preventive manner such as the roll-out of MenAfriVac, or in reactive campaigns during epidemics) and antibiotic prophylaxis. Recommendations for the use of these two strategies vary greatly depending on context.

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In the African meningitis belt, the emphasis has historically been on reactive mass vaccination campaigns. Unfortunately, because these campaigns can be difficult to organize, and because epidemics due to NmA were often explosive and short-lived, the impact of such campaigns has been called into question [10, 11]. Shortening the delay between epidemic declaration and vaccination could potentially have an important impact , but logistical and operational hurdles are difficult to overcome in many contexts.

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On the other hand, antibiotic chemoprophylaxis was implemented in the pre-vaccine era in sub-Saharan Africa and is commonly used among household contacts of cases during outbreaks in western countries [10, 13], but it is not recommended as part of the usual response to meningitis outbreaks in sub-Saharan Africa . This recommendation has been made based on expert opinion that implementing prophylaxis would divert efforts from case management and reactive vaccination campaigns. Nonetheless, data from the 2015 epidemic in Niger suggested that attack rates for household contacts were approximately 20 times higher than that of the general community, suggesting that this group could be an important target for prevention activities . On the other hand, in the same epidemic, 86% of households reported only one case, which could mean that the public health impact of targeting only household contacts could be limited. A recent meta-analysis of four small studies showed a substantial reduction in subsequent cases of meningitis in households receiving chemoprophylaxis . Three antibiotic regimens have been considered as a prophylaxis in other settings: single-dose oral ciprofloxacin, single-dose injectable ceftriaxone, and a 2-day, four-dose course of oral rifampicin. The logistical problems inherent with using an injectable drug like ceftriaxone on a large scale and the unavailability of rifampicin as a single drug in countries with a high prevalence of tuberculosis make ciprofloxacin the best choice for potential prophylactic use in meningococcal meningitis outbreaks in Africa.

review

Because of the threat from NmC and the relative scarcity of NmC-containing vaccine for the foreseeable future, an expert group convened by the World Health Organization (WHO) called for a clinical trial to document the effectiveness of the use of ciprofloxacin chemoprophylaxis as an epidemic response in sub-Saharan Africa . Given that one concern about the widespread use of ciprofloxacin would be the development of fluoroquinolone-resistant bacteria (particularly in the gastrointestinal tract), we will carry out a parallel sub-study to assess the community-wide prevalence of ciprofloxacin-resistant enterobacteriaceae over time.

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We have designed a cluster-randomized trial in response to this call. The proposed trial surpasses the original recommendation of household-level chemoprophylaxis by adding an additional evaluation of village-level chemoprophylaxis, which may be easier to implement and more effective in an epidemic context. Cluster-randomized trials of preventive interventions for both infectious and non-infectious diseases have been implemented in many different African settings [17–20]. We have also designed a sub-study to respond to one of the caveats to this call for a trial, which will investigate the effect of ciprofloxacin chemoprophylaxis on the selection of antimicrobial resistance.

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The study is designed as a cluster-randomized trial with three parallel arms in the setting of a future meningitis epidemic. The primary objective is to compare the incidence of meningitis among villages receiving standard care, household single-dose ciprofloxacin prophylaxis, and village-wide single-dose ciprofloxacin prophylaxis. Secondary objectives include comparing the incidence of meningitis between villages in the three arms by sex and age.

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The study will be implemented in a Health District (HD) experiencing a meningitis outbreak. In order to launch the study protocol, at least two Health Zones (HZs) of the HD will have met the weekly meningitis epidemic threshold . All villages in HZs included in the study will be eligible for inclusion. A village will be randomized to a study arm and receive its intervention once the first case has been reported from that village after the study has opened in an HZ. On inclusion in the study, a dedicated study nurse and at least two dedicated community health agents who have been trained in study procedures and case definitions will be stationed in each HZ.

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Because it is impossible to predict which villages will notify cases in advance of an epidemic, the randomization list will not be prepared in advance, so as to ensure a balanced randomization. Villages will be randomized in a 1:1:1 ratio to receive standard care, household prophylaxis or community-wide prophylaxis.

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Once an HZ has met inclusion criteria, a set of sequentially numbered, sealed envelopes will be prepared, each containing the name of an intervention arm. When the first case presents from a village, study staff in the Health Center (HC) will telephone a central randomizer, who will be otherwise independent of the study functioning.

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The central randomizer will keep a master list of names of villages in each HZ and their study assignments. A reference list of village names and study assignments will be updated in each HC as villages are included. When a case presents, study staff based in the HC will verify whether the case-patient’s village of origin has already been randomized. This information will in turn be double-checked against the central-level randomization list.

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In the standard care arm, after the notification of the first case from a village, a study nurse will visit the village and lead an informational session focusing on the signs and symptoms of meningitis, as well as the urgency of presenting to the nearest HC if any of those symptoms occur.

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In the household-level prophylaxis arm, each time a case is notified, a study nurse will visit the case’s household within 24 hours. A household is defined as a group of people living in the same building or group of adjacent buildings and under the authority of a single head of household. In the case of polygamy, all wives (and their children) of a male head of household will be considered to be members of a single household, provided that they live in the same building or group of adjacent buildings. In the case of multiple heads of household living in one compound of houses, only persons living under the authority of the head of household of the declared meningitis case will be considered a household member and therefore invited to take ciprofloxacin. The nurse will offer ciprofloxacin to all persons currently living in the household and present at the time of the visit, and supervise intake if they accept. (Household members who have accompanied the notified patient will be offered ciprofloxacin at the HC.) Return visits will be arranged for absent household members. In villages randomized to this arm, prophylaxis of a reported case-patient’s household will occur with each subsequent case notified from that village.

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In the community-wide prophylaxis arm, after notification of the first case, a study nurse will visit the village within 24 hours and meet with community leaders. During this visit, a community-wide distribution of ciprofloxacin will be arranged within 72 hours of the initial case presentation at the HC. These distributions will be organized with local authorities, and they will take place after a series of community meetings explaining the study and the distribution to villagers. In this arm, distribution of ciprofloxacin will occur only once, after the first notified case-patient in a village.

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A single dose of oral ciprofloxacin will be administered using age-based dosing (see Table 1). All doses will be directly observed. Tablets will be administered with water. Children unable to swallow pills will have the tablet crushed and dissolved in sweetened water.Table 1Age-based dosing of ciprofloxacinAgeDose (mg)Formulation≥12 years5001 tablet5–11 years2501 tablet1–4 years125½ tablet (250 mg tablet)3–11 months1002 ml oral suspension (250 mg/5 ml)<3 months751.5 ml oral suspension (250 mg/5 ml)

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Throughout the study period, the diagnosis and treatment of meningitis (including referral) will be provided free of cost throughout the study area. Diagnosis and treatment will follow national protocols. Participation in the study will not affect the eligibility of a village or an HZ for reactive vaccination.

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The following case definitions will be used:Suspected case: Abrupt fever (>38.5 °C rectal or >38.0 °C axillary) and at least one of the following signs: neck stiffness, floppy neck, bulging fontanelle, convulsions, or other meningeal signsConfirmed case: Isolation or identification of a causative organism (Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae serotype B) in the cerebrospinal fluid (CSF) of a suspect case by culture, polymerase chain reaction (PCR), or agglutination test

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Confirmed case: Isolation or identification of a causative organism (Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae serotype B) in the cerebrospinal fluid (CSF) of a suspect case by culture, polymerase chain reaction (PCR), or agglutination test

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Demographic and clinical information will be collected for each suspected case, and a unique identifier will be assigned following national protocols. The village of residence will be directly confirmed with the patient or their caregivers. Information about receipt of ciprofloxacin will also be collected.

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Lumbar puncture and CSF analysis will occur following standard protocols and procedures. Any information collected as a result of this procedure will enter into the national surveillance system and will be crossed with local databases. In addition, in order to obtain accurate denominators for the calculation of attack rates, a team of community health workers will perform a census in each village included in the study.

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Meningitis incidence over the course of the epidemic will be expressed as an attack rate (proportion of the population developing meningitis over the defined time period). The sample size needed to show a difference in the reduction of the meningitis attack rate depends on multiple factors, some of which can be reliably estimated and others which cannot be reliably estimated in advance (distribution of village sizes in the study zone, overall amplitude of epidemic) .

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Additional file 2 presents simulations of the number of clusters necessary given specified attack rates and impact of the ciprofloxacin intervention, assuming an alpha (α) error of 5%, 90% power, and an inter-cluster correlation coefficient of 0.025 [22, 23]. The most likely parameters would need less than 75 villages per arm, which is historically a reasonable number of villages affected in meningitis outbreaks in HDs in Niger and in neighboring countries in the meningitis belt.

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A provisional target sample size will be set, but because of these multiple uncertainties, the final target sample size will be set after four weeks of accumulated study data. This period would be sufficiently long to allow for a description of the demographic co-factors in the study area. Additional file 2 shows high variability depending on these co-factors, but the most likely scenarios present sample sizes that are logistically feasible.

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A total of 200 participants in the standard care arm and 200 participants in the community-level prophylaxis arm will be enrolled in the resistance sub-study. Three stool samples will be collected from each participant, at days 0, 7, and 28 using standard operating procedures (SOPs) developed for stool sample collection in Niger. Samples from day 0 in the community-level prophylaxis arm will be collected prior to distribution of ciprofloxacin.

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Assuming baseline prevalence of ciprofloxacin-resistant enterobacteriaceae of 20%, a sample size of 131 persons in each of the two arms would have 90% power to detect a change in the prevalence to 30% over the three repeated measures. Assuming an attrition rate of 25% and 20% of improperly collected samples, a sample size of 200 persons per arm would allow for the calculation of the primary resistance objective (community-wide prevalence of ciprofloxacin-resistant enterobacteriaceae) with sufficient power.

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Once the resistance sub-study has begun, the first odd-numbered village which notifies a case each day in each of the two arms will be included in the sub-study. An exhaustive list of households will be prepared in these villages, and 20 households will be randomly selected from this list. One member of each household will be randomly selected and will be invited to participate in the resistance sub-study. Only participants who know in advance that they will be absent during follow-up visits will be excluded. If this is the case, a second member of the household will be randomly selected as a replacement.

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A stool sample container will be distributed to each participant the day before sample collection, together with instructions for collecting the sample the following morning. Study staff will then return to the village to collect the fresh stool samples. The same procedures will be followed on days 7 and 28. In villages receiving ciprofloxacin prophylaxis, day 0 samples will be collected up to the time of ciprofloxacin administration in the village. At days 7 and 28, samples will also be accepted the day following the scheduled sample collection (i.e., days 8 and 29), if the participant is not able to provide a sample on the scheduled day.

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The processing of stool samples will depend on the location of the study and will be detailed in the study SOPs. In brief, stool samples will be collected by study staff and either stored at 4 °C or inoculated in a transport medium until reception in the central laboratory. Stool samples will then be plated on a MacConkey agar plate containing ciprofloxacin. After incubation, colonies will be identified using standard microbiological methods. The minimum inhibitory concentration of ciprofloxacin and third-generation cephalosporins will be determined , and 10% of samples will be sent to a reference laboratory for quality control purposes.

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The primary analysis, pairwise comparisons of attack rates, will be made between each intervention group using the Student’s t test, weighted to account for varying cluster size, and following an intention-to-treat basis. If the assumption of normality does not hold, we will consider a transformation of the data or a non-parametric test such as the Wilcoxon rank-sum test. Attack rates in each arm will be expressed as a weighted mean of cluster-specific attack rates, and will be presented with their 95% confidence intervals. Each of the two intervention groups will be independently compared to the standard care arm. The overall effect of prophylaxis in the intervention arms will be calculated as OEP = (AR0 – AR1)/AR0, where AR0 and AR1 are the weighted average attack rates in the control and treatment arm respectively . The direct effect of ciprofloxacin prophylaxis will be estimated by comparing attack rates among persons receiving ciprofloxacin versus those not receiving ciprofloxacin in the household-prophylaxis arm.

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The study will take place during an epidemic, and events beyond the control of the study sponsor could affect the propagation of the meningitis epidemic. If major events that could affect the course of the epidemic occur (early rains, a vaccination campaign, etc.), a binary variable (before/after the event) will be added to the data and controlled for using a cluster-level regression model.

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The analysis of the resistance sub-study will be done on the basis of the per-protocol population for whom all three stool samples are available. The primary sub-study outcome (change in community-wide prevalence of carriers of ciprofloxacin-resistant enterobacteriaceae) will be compared using the method described by Liu and Wu . Individual-level acquisition rates at 7 and 28 days following the initial sample will also be calculated with corresponding 95% confidence intervals using the binomial distribution. Acquisition will be defined as the presence of ciprofloxacin-resistant enterobacteriaceae at day 7 or day 28 in a participant who did not have ciprofloxacin-resistant enterobacteriaceae at the time of baseline sample collection. A Fisher exact test will be used to compare proportion of acquisition between study interventions. All tests will be two-sided with a significance threshold of α = 0.05.Due to the uncertainty of several parameters used in the sample size calculation, we plan to recruit for four weeks and perform a sample size re-estimation based on the estimated value of p 0, the attack rate in the controls, from this sub-sample. For this, we follow the method of Lake et al. . We make two assumptions in doing this: that epidemics in individual villages last only a short time, meaning the p 0 estimated 4–6 weeks after recruitment of a village will be very close to p 0 for that village at the end of the study; and that the attack rate in each village will remain constant during the whole course of the epidemic, i.e., that villages affected early in the epidemic do not have systematically bigger or smaller epidemics than villages affected later in the epidemic.

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An SAE is defined as any untoward medical occurrence that, at any dose of ciprofloxacin received, results in death, is life threatening, requires inpatient hospitalization or prolongation of existing hospitalization, or results in persistent or significant disability or incapacity, or any medically important event/reaction that may jeopardize the subject and may require medical or surgical intervention to prevent one of the outcomes listed above.

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Procedures for obtaining informed consent have been developed in accordance with the Ottawa Statement on the Ethical Design and Conduct of Cluster Randomized Trials . Gatekeepers have been defined as the health care professional in charge of the HC and village chiefs and their deputies. Village chiefs will be asked to provide written permission for the randomization of their village. It will not be possible to obtain individual-level informed consent for participation, as each arm will likely include several thousand individuals during an emergency. We have met established criteria for the waiver of individual consent in a cluster-randomized trial [28, 29]. Nonetheless, in villages receiving household chemoprophylaxis, the study nurse will present the risks and benefits of taking ciprofloxacin to household members, and it will be administered only to those who are willing. In the village-wide prophylaxis arm, a series of community meetings will be held in advance of the mass distribution, during which the study and its risks and benefits will be presented and questions may be answered. Those persons who present to the ciprofloxacin distribution site will be considered to have been duly informed of the risks and benefits of taking ciprofloxacin and the voluntary nature of their participation.

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The trial was developed by Epicentre with the support of Médecins Sans Frontières. The primary trial sponsor is Epicentre, who will hold the data and conduct all analyses. The primary sponsor will independently monitor study execution at field sites. A scientific committee has been convened to review drafts of the protocol and analytic plan, and will be consulted for queries regarding implementation and analysis during and after the trial.

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When the trial report is completed, the investigators will share the summary results with local, regional, and national health authorities. The findings from this study will also be published in a peer-reviewed scientific journal and disseminated at appropriate conferences. The ultimate decision to submit a manuscript will remain with the primary sponsor.

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The research data will be the property of the sponsors, but data will be made as widely and freely available as possible, while safeguarding the privacy of participants and protecting confidential data. A de-identified dataset can be made available under a data-sharing agreement that provides for a commitment to using the data only for research purposes and securing data using appropriate technology.

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Despite the undeniable success of MenAfriVac, the emergence of NmC as a potential cause of major epidemics in the African meningitis belt has brought new problems. Limited supplies of vaccine against NmC, combined with limitations inherent to organizing mass vaccinations (particularly regarding their timing), have led to a recommendation to assess the use of chemoprophylaxis among household members as an alternative epidemic response.

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The proposed trial responds to this call, and goes one step further by adding a third arm in which chemoprophylaxis would happen at a village level following the notification of the village’s first case. This addition responds to on-the-field realities in many parts of the meningitis belt, where it may be politically difficult to treat only one household in a small village when other villagers would also be at risk. Also, despite the higher risk for household members, most households will still report only one case during an epidemic, so this community-wide strategy may have a greater impact on overall meningitis incidence.

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On the other hand, given the growing problem of antibiotic resistance, even if this trial were to show overwhelmingly positive results, it would be impossible to recommend chemoprophylaxis as a routine epidemic response without having evidence on the possible effect of mass distributions of ciprofloxacin on the prevalence of resistant pathogens. We believe that by carrying out the sub-study during the epidemic we will provide the most realistic evidence on which to base potential decision making.

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All resources, materials, and authorizations necessary for the trial are in place in Niger, and surveillance is ongoing to detect eligible districts during upcoming meningitis epidemics. We are currently considering submitting the protocol in other meningitis belt countries.

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Additional file 1:Populated SPIRIT checklist. These are our responses to the standard SPIRIT checklist. (DOC 121 kb) Additional file 2:Sample sizes necessary for the trial given different conditions. This table presents a range of different possibilities for sample size necessary for the trial, depending on various conditions, some of which will not be known until after the trial’s start. (XLSX 22 kb)

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Sample sizes necessary for the trial given different conditions. This table presents a range of different possibilities for sample size necessary for the trial, depending on various conditions, some of which will not be known until after the trial’s start. (XLSX 22 kb)

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In 2016, 1.3 million people died from tuberculosis (TB) and 1 million people died from HIV. Of the 10.4 million people that develop TB each year, one out of ten is HIV positive . HIV is one of the most important challenges to TB control globally . By severely affecting the immune system, HIV facilitates TB dissemination and increases mortality of coinfected persons as compared to TB patients who are HIV negative [2, 3]. Active TB is an AIDS-defining disease and a criterion to start antiretroviral therapy (ART); therefore, diagnosis of both diseases is important in co-infected patients [4–8].

review

The optimal HIV screening strategy has been debated since the availability of HIV screening tests in 1985. Initially, HIV screening was mainly done in blood banks. In 1987, counseling and screening was recommended to persons at risk of HIV infection such as those with risk behaviors and those seeking care for sexually transmitted infections (STI). In 1993, screening was extended to patients attending hospitals that reported an HIV prevalence above 1% among their patients. In 1995, learning that zidovudine could prevent HIV vertical transmission prompted the implementation of universal screening among pregnant women . Despite these efforts, a low proportion of persons infected with HIV were aware of their status, and a high proportion of patients were diagnosed in late stages of HIV infection. To increase HIV diagnosis, a voluntary counseling and testing strategy required scaling up . In 2005, the Center for Disease Control (CDC), recommended provider-initiated counseling and testing to all persons between 13 and 64 years unless the person opts out [9, 11]. The World Health Organization (WHO) recommends HIV voluntary counseling and testing as a standard practice among TB patients to increase HIV status awareness . However, in 2016, the WHO estimated that 57% of notified cases of TB were aware of their HIV status .

review

In Peru, HIV is concentrated among high-risk groups and 0.4% of the general population is infected, on the other hand, TB burden is among the highest in the region: in 2016 the incidence for all TB cases was 117 per 100,000 population . As the burden of TB is higher, case detection and treatment is conducted in the first level of health care. Conversely, because of its low prevalence, HIV/AIDS is managed in referral centers . TB nurses working in peripheral health facilities offer voluntary counseling and testing for HIV of TB patients routinely. Peru adopted the HIV voluntary counseling and testing strategy among TB patients in 2006 . The WHO reported that of the 31,079 TB patients notified in 2016, 84% were screened for HIV. Of them, 6% were HIV positive . TB-HIV co-infection is a challenge for public health, both for diagnosis and timely management and for the prognosis of both diseases. It is essential to expand HIV screening of TB patients and timely initiation of ART to improve individual prognosis, to reduce HIV transmission and to reduce the burden of HIV and TB. This study aims to determine the proportion of newly diagnosed TB patients screened for HIV in a district with high TB incidence in Lima, Peru and to analyze patient’s and health system’s characteristics associated to not being screened for HIV.

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We conducted a secondary data analysis of cross sectional data obtained within a cohort study in 34 health care facilities (one district hospital and 33 primary care health centers) managed by the Ministry of Health in the San Juan de Lurigancho district in Lima, Peru. This was a cohort study to determine the proportion of drug resistant TB and the rate of recurrent episodes among TB patients. A sample size of 1130 was calculated for those objectives. Adults with a smear-positive pulmonary TB episode diagnosed between March 2010 and December 2011 with no history of previous TB treatment were enrolled. Patients initiated anti-TB treatment at the TB clinic and were followed at the health facility until the end of their treatment regimen. Treatment outcomes were prospectively obtained from the NTP registers. TB registers were monitored monthly up to two years after the end of treatment of the last enrolled case for TB recurrence. If a recurrent episode was found among an enrolled and cured TB patient, a sputum sample was collected [15, 16]. San Juan de Lurigancho is a periurban district in the north east of Lima and lies on the side of a hill. It is the most populous district in Peru, with over one million inhabitants. In 2015, TB incidence in Lima was 164.9 per 100,000 inhabitants .

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The NTP guidelines recommend that all TB patients should offered HIV screening with an ELISA or a rapid test after a pre test counseling session conducted by a TB nurse trained by the HIV program. A post test counseling session is given in referral health facilities where HIV multidisciplinary teams provide HIV care .

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The cohort study included consenting adult patients with a first episode of smear positive pulmonary TB diagnosed between March 2010 and December 2011 in one of the study sites. Patients that had received more than two doses of TB treatment were excluded. This secondary data analysis included all cohort participants.

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Trained field workers enrolled participants and applied a structured questionnaire to collect demographic, epidemiological and clinical data. The variables sex, age, weight loss, education, social economical status, marital status, illegal drug consumption, alcohol abuse, history of deprivation of liberty, history of diabetes mellitus, employment and place of birth were obtained through face to face interviews while smear, culture and drug susceptibility test (DST) results, treatment outcomes, and HIV test results were retrieved from clinical files. A single sputum specimen was collected from each participant at enrollment. Sputa were transported to the Tuberculosis Laboratory of the Instituto de Medicina Tropical Alexander von Humboldt analyzed under smear microscopy and cultured on Löwenstein-Jensen media. The proportion methods evaluated drug susceptibility to isoniazid, rifampicin and ethambutol. Results were reported to the health facility [15, 16]. The Tuberculosis Laboratory at the Instituto de Medicina Tropical Alexander von Humboldt conducted regular quality control assurance procedures for smear microscopy, culture and DST.

study

Study participants that were not yet screened for HIV by the TB routine staff at the moment of the interview, were invited for screening. Those agreeing to be screened received counseling by the Ministry of Health designated staff and the study field workers drew a sample of blood. An ELISA or a rapid test was done. If positive, the diagnosis of HIV infection requires a confirmatory test (Indirect Immunofluorescence-IFI or Western Blot). ELISA for HIV was done at the Instituto de Medicina Tropical Alexander von Humboldt. Results were given to the TB staff that delivered the results to patients and managed them accordingly. Patients with a positive test (either obtained by the Ministry of Health routine procedures or by the study) were referred to HIV services (in Peru, these are located in referral hospitals) for a confirmatory test and post test counseling. If HIV was confirmed they were enrolled in the Ministry of Health HIV program where ART is provided free of cost to patients. The criteria to initiate ART at the time of the study (2010–2011) were: Any symptomatic HIV-positive person with clinical stage B or C regardless CD4 count, any HIV-positive person with a CD4 count of 200 cells / mm3 regardless of clinical stage and other criteria determined by an expert committee. Clinical files of all TB-HIV positive patients were reviewed at the end of their TB treatment. We registered the date in which they were enrolled in the HIV program at the referral facility and the date of ART initiation.

study

For the cohort study logistics, the district was divided in four geographical areas depending on the altitude related to the hill of the study district and balancing the number and size of facilities in each area: highest, high, middle and lower. A field worker was assigned to each area. The two higher areas lie by the side of the hill that is less urbanized with poorer housing quality, and the middle area and lower areas, are on the flatter and more urban area of the district.

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Data were entered in a specially designed database in Access for Microsoft Office (Microsoft Corporation, Redmond, WA, US) and analyzed using Epi Info ™ version 7.1. The outcome was HIV screening (done or not done). Patients not screened were further categorized in “opted out” which included participants that explicitly opted out of HIV screening by saying “no, I do not want to be screened” and in “screening not done for unknown reasons” which included participants that did not explicitly opted out, but either HIV screening was not offered for unknown reasons or despite it being offered, it was not done for unknown reasons. This categorization aimed to compare the characteristics of patients opting-out to those who did not explicitly opted out but screening not done for diverse reasons (patient may not want to be screened but does not say so, or he/she may want but it is not done, because of lack of follow up by the staff or other reasons). The hypothesis underlying this subclassification was that TB patients opting out of HIV screening may have a higher risk of HIV infection and fear of it makes them opt out. While this hypothesis cannot be confirmed in this study, a comparison of patients opting out with those not screened without explicitly opting out, could be relevant. Age was categorized in young adults (18–34 years), adults (35–59 years) and older adults (60–90 years). The 22-months study period was divided in four periods to determine time trends in HIV screening. HIV screening could be different in time if there were screening tests stock outs, or if staff changes in time affected performance related to HIV screening. Socioeconomic status was measured by a Peruvian Ministry of Finance validated scale and categorized patients in poor (including extreme and not extreme poverty) and not poor . Alcohol consumption was measured using the validated CAGE four-question screening test . Those that had a high suspicion of alcoholism we classified as alcoholics.

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