We tested the hypothesis that activation of the innate immune response induces an imbalance in the proteolytic homeostasis in the peripheral airways of healthy subjects, towards excess serine or gelatinase proteinase activity. During bronchoscopy, 18 healthy human subjects underwent intra-bronchial exposure to endotoxin and contra-lateral exposure to vehicle. Bronchoalveolar lavage (BAL) samples were harvested 24 or 48 hours (h) later. We quantified archetype proteinases, anti-proteinases, inflammatory BAL cells, and, importantly, total plus net proteinase activities using functional substrate assays. As expected, endotoxin exposure increased the concentrations of polymorphonuclear leukocytes (PMN's) and macrophages, of proteinases and the anti-proteinases tissue inhibitor of metalloproteinase-1, α-1-antitrypsin and, to a lesser extent, secretory leukoproteinase inhibitor, at both time points. Notably, at these time points, endotoxin exposure substantially increased the quantitative NE/SLPI ratio and the net serine proteinase activity corresponding to neutrophil elastase (NE). Endotoxin exposure also increased the total gelatinase activity corresponding to matrix metalloproteinase (MMP)-9; an activity dominating over that of MMP-2. However, endotoxin exposure had no impact on net gelatinolytic activity at 24 or 48 h after exposure. Thus, local activation of the innate immune response induces an imbalance towards increased net serine proteinase activity in the proteolytic homeostasis of the peripheral airways in healthy subjects. Hypothetically, this serine proteinase activity can contribute to tissue remodelling and hypersecretion via NE from PMN's, if it is triggered repeatedly, as might be the case in chronic inflammatory airway disorders. A well-maintained balance between the local serine proteinases, gelatinases and their inhibitors is believed to be critical for preserving tissue structure and function in the airways, as well as for coordinating the collective immune responses from structural and inflammatory cells to maintain host defence It is known that both polymorphonuclear leukocytes (PMNs) and macrophages are important producers of proteinases and matching anti-proteinases Even though there is accumulating evidence that the maintenance of the proteolytic homeostasis is involved and altered in several chronic airway disorders, surprisingly little is known about more holistic and functional aspects of the proteolytic homeostasis in the airways of healthy human subjects In the current study, we tested the hypothesis that activation of the innate immune response induces an imbalance in the proteolytic homeostasis in the peripheral airways, towards excess serine proteinase or gelatinase activity. To test this hypothesis, we conducted a functional plus a quantitative characterization of the proteolytic homeostasis with and without local exposure to the toll-like receptor (TLR)-4 agonist endotoxin. Thus, endotoxin was instilled in a segmental bronchus and its vehicle solution in a contra-lateral one, within each healthy volunteer examined All participating subjects gave oral and written informed consent, in accordance with the Helsinki Declaration. The appropriate ethical permission  was obtained from the Ethical Review Board for studies on humans in Gothenburg.Our study was performed at the Clinical Section of Respiratory Medicine at Sahlgrenska University Hospital in Gothenburg, Sweden. Healthy human volunteers were recruited through local advertising. The clinical part of the study took place from Nov 11, 2003 to Jan 14, 2005. All samples were processed in the involved laboratories during 2006 to 2008.During the first clinical visit, the medical history was recorded and physical examination, spirometry and electrocardiogram were performed. Current or past smoking, a history of allergy or atopy and any regular medication constituted exclusion criteria, with the exception of oral contraceptives.1) >80% of predicted value, as well as normal findings in their electrocardiogram and physical status. Bronchoscopies were performed during the second and third visit, separated by an interval of either 24 or 48 h. The allocation of subjects to either time point was performed on a practical basis, set by the complex hospital logistics. Endotoxin exposure took place at visit two  and BAL was conducted at visit three .All included subjects had a normal ventilatory lung capacity defined as forced expiratory volume during one second  for analysis of haemoglobin, total blood leucocytes (LPC) and differential leukocyte counts as well as C-reactive protein (CRP). Plasma and serum from additional blood samples was separated, frozen and stored at −70°C pending further analyses.All bronchoscopies were performed trans-orally by one very experienced bronchoscopist who is a board-certified Clinical Specialist in Respiratory Medicine  with the subject in supine position. We utilized a slightly modified version of a previously published endotoxin exposure protocol Eschericia coli 0113: H10, from USP, Rockville, MD, USA) and inflation of air (10 mL). Endotoxin was given in a dose proven both efficacious and clinically safe (4 ng/kg diluted with PBS up to 10 mL of fluid) The following procedures were applied for B.I: A balloon-tipped catheter was inserted through the bronchoscope, placed in a bronchial segment (either right middle lobe or lingula) and inflated with air to seal off the chosen segment proximally before challenge. The instillation of vehicle  and inflation of a small volume of air (10 mL) into the bronchial segment was then conducted. The bronchoscope was subsequently retracted and transferred to the corresponding segment in the contra-lateral lung. The sealing off-procedure was followed by instillation of endotoxin , starting with the vehicle-exposed segment. After the subsequent aspiration of each 50 mL aliquot, the entire harvested yield was pooled in a siliconized glass container. This BAL sample was immediately transported on ice to our research laboratory. The recovered volume was then measured and the fluid filtered for retention of mucus and cell debris. After this, the filtered BAL sample was centrifuged . The cell-free BAL fluid (i.e. the centrifugation supernatant) was then separated and frozen at –80°C until further analysis.The cell pellet from the centrifugation was re-suspended in PBS and cell viability was determined using trypan blue exclusion as measure. The total cell count was determined utilizing a haemocytometer (Bürker chamber). The differential counts for BAL cells were performed utlizing stained cytospin preparations after counting of 2×300 cells using a conventional light microscope .Clinical symptoms were recorded immediately prior to B.I and B.II, and at three different time points after each bronchoscopy, using a questionnaire (supporting information).2 fragment donkey anti-goat IgG (Jackson ImmunoResearch™ laboratories Inc) was used, followed by alkaline phosphatase-conjugated streptavidin (DAKO). Bound antibodies were visualised with Vector Red Alkaline Substrate Kit . Mayer's Hematoxylin  was used for counter-staining. The stained ICC samples were assessed in a blinded fashion under a light microscope (Axioplan® 2) at a magnification of ×400.To prepare for immunocytochemistry (ICC), the BAL cells were fixed  and washed twice in buffer prior to making the cytospin preparations. Air dried samples were stored in –83°C until further use. To avoid unspecific binding, samples were treated with 10% donkey serum . Endogenous biotin was blocked using Biotin Blocking System . The ICC slides were further incubated with a polyclonal goat anti-human MMP-9 antibody  during 1 h. As secondary antibody, a biotinylated F(ab‚)Examining cell-free BAL fluid, we quantified the archetype gelatinases matrix metallo-proteinase (MMP)-2 and MMP-9 When analysing cell-free BAL fluid, we also quantified the concentrations of endogenous anti-proteinases including tissue inhibitor of metalloproteinase (TIMP-1), that binds and inactivates MMP-9 These measurements were based upon functional substrate assays. The net gelatinase activity in BAL fluid (BALF) was measured as previously described The net serine proteinase activity was measured as previously described p-values <0.05 were accepted as statistically significant. Repeated comparisons were not compensated for, to avoid an overly conservative outcome of the analysis.The SPSS 14.0  software package was used for the statistical analyses. Since normal distribution of results could not be proven, due to the limited sample sizes, data are presented in a non-parametric manner, as median and individual values with range. For comparisons between groups, paired (Wilcoxon Signed rank test) or unpaired (Mann Whitney U-test) non-parametric tests were used. As appropriate, paired comparisons were used for matched samples obtained from the same subjects . Unpaired comparisons of the effect of endotoxin exposure over time (samples from different subjects) were made by calculating the difference between results from the vehicle- and endotoxin-exposed segment within each subject, at each time point, followed by a comparison of these differences between 24 and 48 h. For correlations between individual data in different groups Spearmańs two-tail rank correlation test was applied. Nineteen healthy human volunteers  completed the study protocol. One female subject in the 48 h group experienced transient nausea and vomited immediately after B.I. Due to the risk of micro-aspiration influencing the BAL yield, this subject was excluded. Consequently, we finally included 11 subjects at 24 h and 7 subjects at 48 h. Symptoms of side effects associated with B.I, including the endotoxin exposure, were mild and transient ×104 cells/mL, p<0.05] and 48 h . This was also true for the concentrations of PMNs in BAL samples   samples , which w samples  and were samples .The zymography analysis identified clearly detectable “bands” of gelatinolytic activity in BAL fluid, regardless of endotoxin exposure, mainly at 72 kD and 92 kD, corresponding to MMP-2 and MMP-9, respectively . There was also a third band at 110-130 kD, corresponding to the size of the complex of MMP-9 and HNL The concentrations of MMP-9, its inhibitor TIMP-1, MMP-2 and HNL are shown in versus 0.36 (0.05–1.26), p = 0.001, n = 11] and at 48 h  after endotoxin exposure.The concentrations of NE, SLPI and AAT are shown in The ICC staining for MMP-9 indicated intracellular localization of this gelatinase in PMNs mainly, at both time points . The proThere was a higher net serine proteinase activity in the BAL fluid from endotoxin- than in the vehicle-exposed segments at 24 and 48 h after exposure . There wnet activity for serine proteinases but not gelatinases.With this study, we tested the hypothesis that activation of the innate immune response induces an imbalance in the proteolytic homeostasis in the peripheral airways. We found that after activation of the innate immune response with the TLR-4 agonist endotoxin, healthy human subjects displays an imbalance in the proteolytic homeostasis in the peripheral airways, towards an increase in the We think that the identification of net serine proteinase activity as a discriminating factor in the peripheral airways was facilitated by the design of our study, including non-smokers only and a protocol with limited and well-defined exposure to endotoxin. The fact that our protocol controlled for vehicle, bronchoscopy and time within each subject is probably important as well.The serine proteinase substrate N-methoxysuccinyl-ala-ala-pro-val-p-nitroanilide is well-documented in the literature On a hypothetical basis, it could be argued that the induced and sustained increase in serine proteinase activity that we demonstrate relates to the dose and route of administration of endotoxin. However, there is published evidence supporting an increase in serine proteinases caused by bacterial stimuli, obtained in studies with different doses and routes of administration. These studies include the utilization of inhaled as well as intra-bronchial administration of endotoxin; the latter as in our current study Notably, our current demonstration of an endotoxin-induced imbalance towards increased net serine proteinase activity may have bearing not only for gram-negative but also for gram-positive infections. This is because local impairment of “anti-neutrophil elastase capacity” has been demonstrated in patients with community-acquired pneumonia, predominantly caused by Streptococcus pneumonie Indeed, the serine proteinase inhibitor AAT was increased within 24 h after endotoxin exposure in our study; the time point associated with the highest net serine proteinase activity. Since BAL samples primarily reflect conditions in the peripheral airways, and presumably the alveoli as well, this observation argues against AAT being sufficient for protecting against excess NE activity at this level As expected, our results confirm that the immune response to endotoxin in the peripheral airways of healthy subjects include a very pronounced accumulation of PMNs and a less pronounced accumulation of macrophages. Whereas the PMN accumulation tended to fade the macrophages peaked at 48 h after exposure. Interestingly, we noted engulfment of seemingly apoptotic PMNs in macrophages at 48h as well, suggesting an effective resolution of the innate immune response, in line with previous studies on induced sputum after exposure to inhaled endotoxin in healthy airways per se, to bronchoscopy (B.I), as well as the response to some “spill-over” of endotoxin from the contra-lateral bronchial segment at each time point. In our study, we thus controlled for vehicle, bronchoscopy and timing within each subject.We think that the fact that there was a decline in BAL PMN concentrations from 24 to 48 h in vehicle-exposed segments, mirroring the endotoxin-exposed segments, can be attributed to our exposure model. In this model, BAL samples from vehicle-exposed bronchial segments reflect the sum of “inherent conditions” plus the “un-specific response” to vehicle The increase in absolute MMP-9 concentrations after endotoxin exposure followed the corresponding increase in PMN and NE concentrations at 24 h after exposure in our study. The same pattern was evident for the ICC signal of MMP-9; a signal predominantly observed in PMNs. However, as opposed to the endotoxin-induced increase in MMP-9 concentrations, the corresponding increase in NE concentrations remained virtually unaltered at 48h. This also indicates a more sustained innate immune response for healthy, non-smoking subjects in terms of NE compared with MMP-9.The concentrations of HNL tended to be higher at 48 h in BAL samples from endotoxin- than from vehicle-exposed segments, a pattern that is clearly different from what we observed for PMNs, MMP-9 and NE. The mechanistic rationale for this observation remains uncertain but may relate to the fact that HNL, MMP-9 and NE all originate from different types of intracellular granulae We found that the time course of alterations in TIMP-1 concentrations relatively well followed that of MMP-9 concentrations, although the concentration of TIMP-1 declined somewhat more than that of MMP-9 at 48 h. This is of course compatible with the proteolytic homeostasis being critical to preserve structure and function of the airways, to avoid the excess proteinase activity present in inflammatory disorders like COPD and CF net gelatinase activity. The net gelatinase activity is likely to be the critical functional assessment, reflecting a balance between gelatinases and the matching anti-proteinases. Possibly, the method (ELISA) that we utilized to detect the endotoxin-induced increase in MMP-9 concentrations does not strictly reflect the finally processed MMP-9; the induced release of pro-MMP-9 molecules that are not enzymatically active may also have been detected. It could also be that the lack of an endotoxin-induced increase in net gelatinase activity reflects physiological conditions rather than pathology. To further evaluate these matters, our current study design should be applied in new clinical studies.Our assessment of total gelatinase activity (using zymography) suggests that MMP-9 is the quantitatively dominant one among archetype gelatinases in the peripheral airways of healthy subjects, with or without activation of the innate immune response. However, our quantification of the absolute MMP-9 concentrations (using ELISA) and the functional assessment of net gelatinase activity  did not verify a clear association of these two outcomes. Unexpectedly, our quantification of net gelatinase activity showed that this functionally decisive outcome was comparable in BAL samples with or without endotoxin exposure. Thus, our results suggest that the net gelatinase activity is relatively stable over time with or without activation of the innate immune response. At a first glance, this finding may appear contradictory to previous speculations that the excess release of MMP-9 is involved in tissue remodelling in severe asthma and COPD net serine proteinase activity may have implications for the development of pharmacotherapy targeting endotoxin-related airway disease. New clinical studies in acute and chronic airway disorders are therefore warranted.In conclusion, the key finding of the current study is that activation if the innate immune response with a TLR-4 agonist induces an imbalance towards increase in net activity of serine proteinases but not gelatinases in the peripheral airways. The utility of this imbalance remains unclear since, hypothetically, it can contribute to pathology in terms of tissue remodelling and destruction, to the stimulation of pro-inflammatory cytokines, other proteinases and gland secretion via NE from PMN's, if it is triggered repeatedly File S1Table S1. Symptom assessment after the first bronchoscopy (B.I) in 18 healthy subjects. n =  number of subjects reporting each symptom. Table S2. Blood concentrations of C-reactive protein (CRP), total leukocytes (LPC) and polymorphonuclear leukocytes (PMN's) at baseline prior to bronchoscopy I (B.I), and 24 or 48 hours (h) after intra-bronchial endotoxin exposure in healthy subjects. Table S3. Recovery of and cell viability in bronchoalveolar lavage (BAL) fluid samples harvested after intra-bronchial exposure to vehicle (Veh) and endotoxin (E), respectively, in 18 healthy subjects. Samples were harvested either 24 (n = 11) or 48 (n = 7) hours after the exposure. Figure S1. Photo of representative zymography gel, based upon analysis of cell-free bronchoalveolar lavage (BAL) fluid samples harvested after intra-bronchial exposure to vehicle (no arrow) and endotoxin (arrow), respectively, at 24 hours  or 48 h (open arrow) in healthy subjects. The samples display three bands representing different molecular weights, corresponding to matrix metalloproteinase (MMP)-2 (72 kD), MMP-9 (92 kD) and a complex bound form of MMP-9 (130 kD). *Two lanes from the same subject. Figure S2. Photomicrograph of representative cytospin slides showing bronchoalveolar lavage (BAL) cells containing intracellular matrix metalloproteinase (MMP)-9 harvested after intra-bronchial exposure to endotoxin and vehicle, respectively, in healthy subjects. Immunoreactivity to MMP-9 is red and polymorphonuclear (PMN) cells with positive staining are indicated with arrows. Panels show samples from segments exposed to a) endotoxin and b) vehicle from the same subject at 24 hours (h) and c) endotoxin at 48 h after exposure from another subject. In panel c, the PMN indicated by an arrow is engulphed by a macrophage.(DOCX)Click here for additional data file.


Evidence from cell studies indicates that persistent organic pollutants (POP) can induce insulin resistance, an essential component of the metabolic syndrome (MetS). We hypothesized that residential proximity to environmental sources of POP would be associated with the MetS in the population. The present study examined the association between residency in a zip code containing or abutting environmental sources of POP and MetS-related hospitalization rates. Hospitalization data were obtained from the New York Statewide Planning and Research Cooperative System. Relative risks (RR) were calculated as hospitalization rate ratios. Adjusted RR and their 95% confidence intervals (CI) were estimated by multivariable Poisson regression. A higher proportion of African Americans resided in POP zip codes compared to Caucasians . Residence in POP zip codes was associated with a statistically significant 39.2% increase in MetS-related hospitalization rates, adjusted for race, gender, and age . Increase in age was independently associated with higher MetS-related hospitalization rates (p for trend < 0.001). Our findings contribute to the body of evidence supporting the hypothesis of POP constituting an environmental risk factor for the MetS. Further studies investigating exposure to POP and insulin resistance are warranted. Age-aThe MetS increases risk of myocardial infarction (heart attack) and stroke; it is also associated with an increased cardiovascular disease mortality and all-cause mortality ,9.-p-dioxins , polychlorinated dibenzofurans, and persistent pesticides, such as dichlorodiphenyltrichloroethane (DDT) [Persistent organic pollutants (POP) are a group of organic compounds that are highly resistant to chemical, photolytic, and biological degradation. These lipophilic compounds are persistent in the environment and human body. POP include polychlorinated biphenyls (PCB), polychlorinated dibenzone (DDT) . PCBs wene (DDT) ,12. Inadne (DDT) . POP arene (DDT) .Recent cell studies reported that POP can interfere with insulin signaling pathway and induce insulin resistance ,16. Resu2.2.1.We conducted a population-based semi-ecological study of MetS-related hospitalizations in relation to presumed environmental exposure to POP, based on zip codes of residence. Semi-ecological designs are used widely in environmental exposure studies. They utilize exposure status measurement on a group level combined with confounder measurement  on an individual level ,20. PatiBoth the principal and secondary diagnoses are coded according to the International Classification of Diseases, the Ninth Revision, Clinical Modification (ICD-9-CM). We used data for a three-year period of 2002–2004 for NYS except for New York City. New York City maintains its own hospitalization database and its population structure is different from the rest of the NYS. MetS-related hospitalizations were identified as hospitalizations containing the ICD-9-CM diagnosis code 277.7 as either the principal or a secondary diagnosis in the hospital discharge record. This ICD-9-CM code for the MetS was approved by the National Center for Health Statistics in 2001 .Patients’ environmental exposure status was designated as “POP” or “non-POP” based on their residence in zip codes containing or abutting hazardous waste sites known to contain dioxins/furan, PCBs or persistent pesticides; details of these classifications were described elsewhere . Zip codData on race, gender, age, and income by zip code were obtained from Claritas, Inc., derived from the US Census. Because patients of the two largest racial groups—Caucasians and African Americans—comprised more than 98% of the residential NYS population exclusive of New York City and accounted for more than 98% of the MetS-associated hospitalizations, other racial groups were not included for the purpose of minimizing extra variability and preserving the parsimony of the model.After the above restrictions were applied, there were a total of 408 MetS-associated hospitalizations among 25-years and older adults during 18,113,583 person-years, over a three-year period of 2002–2004.2.2.The primary outcome variable was the MetS-associated hospitalization rate. Hospitalization rates  were calculated as a number of MetS-associated hospitalizations divided by the population residing in a given zip code multiplied by 100,000.Relative risks (RR) of MetS-associated hospitalization in relation to POP exposure status were calculated as hospitalization rate ratios. The multivariable Poisson regression was utilized to obtain RR of MetS-associated hospitalization, with respective 95% confidence intervals (95% CI). Multivariable adjusted analysis was used to control for confounding by age, race, and gender by including the respective variables into the multivariable model.All statistical analyses were performed with SAS software, version 9.2 . The PROC GENMOD procedure was used to conduct the multivariable Poisson regression. To adjust for overdispersion, a scaling factor was used; the scaled Pearson chi-square was equal 1. A conventional p < 0.05 value  was used for all analyses.3.3.1.The mean ± SD age of the patients was 58.9 ± 14.4 years. Almost 55% of hospitalized patients were women (224 cases) and about 45% were men (184 cases). Of the total of 408 MetS-associated hospitalizations over a three-year period, 16 (4%) were African American and 392 (96%) Caucasian, which reflects the fact that African Americans constitute 6% and Caucasians 94% of the study population. Both unadjusted (not controlled for confounding) and adjusted (controlled for confounding) analyses were conducted. Unadjusted analyses provided preliminary information on the distribution of MetS-associated hospitalizations across different race, age, and gender groups. Adjusted analyses were conducted afterwards for the purpose of investigating whether an association between residence in a POP zip code and MetS-associated hospitalizations exists.3.2.While unadjusted MetS-associated hospitalization rates were lower in African Americans than in Caucasians, the difference was not statistically significant  . UnadjusUnadjusted analysis of MetS-associated hospitalizations in relation to age indicated a statistically significant increase of hospitalization rates with age . DiffereStratified unadjusted analyses, comparing MetS-associated hospitalization rates in the populations of POP and non-POP zip codes, were conducted separately for race, gender, and age categories . Both AfThe two different patterns of MetS-associated hospitalizations by age described above for the entire study population were also found in a stratified analysis. Hospitalization rates in the two youngest categories were below the respective POP and non-POP average hospitalization rates , while for the ages of 45 years and older they were above average  for resi3.3.While unadjusted analysis provides useful information on the distribution of hospitalization rates across different population groups, it cannot be used for making conclusions on whether hospitalization rates are associated with exposure, because the results are not controlled for confounding. To investigate whether residence in POP zip codes is associated with an increase in MetS-associated hospitalization rates after controlling (adjusting) for race, gender and age, we conducted a multivariable adjusted analysis, using a multivariable Poisson regression model.Results of the multivariable Poisson regression analysis are presented in 4.In this study we found that residence in zip codes containing or abutting environmental sources of POP is associated with a statistically significant 39.2% increase in MetS-related hospitalizations compared to residents of non-POP zip codes. To our knowledge, this is the first study investigating the association between the presumed environmental exposure to POP based on residence near to a POP hazardous waste site and MetS employing hospitalization rate as the outcome of interest.We chose to focus on this outcome for two reasons. Firstly, while an increased MetS-related hospitalization rate does not prove a causal effect of POP, it is reasonable to expect that the hypothesized association between POP and MetS does exist. Our previous studies using hospitalization in relation to residence near to POP sites have reported elevated risk of diabetes , hyperteet al. (2011) investigated insulin resistance in non-diabetic adults residing near a deserted pentachlorophenol factory that generated polychlorinated dibenzo-p-dioxins and furans as byproducts. They found a statistically significant positive association between serum dioxin levels and homeostasis model assessment—insulin resistance , fasting glucose, systolic and diastolic blood pressure, waist circumference, and HDL cholesterol level [Our results are consistent with findings from other population-based studies of exposure to POP and MetS, or MetS components, that were conduced using different approaches. Chang ol level .et al. (2000) studied association between environmental residential exposure to TCDD and insulin levels in 69 subjects with normal glucose tolerance test who lived within 25 miles of a Superfund site in Arkansas [Cranmer Arkansas . The souet al. (2007) investigated cross-sectional associations between POP and MetS among 721 adults, of whom 175 individuals had MetS [-p-dioxins) were found to be associated with only one of the MetS components—HTN.Using the National Health and Nutrition Examination Survey data, Lee et al. (2009) found that body burden levels of polychlorinated dibenzofurans, polychlorinated dibenzo-p-dioxins, and dioxin-like PCBs were statistically significantly associated with the prevalence of the MetS [In a study of 160 MetS patients among 1,374 Japanese residents non-occupationally exposed to POP, Uemura the MetS .-p-dioxin (TCDD) has been shown to stimulate TNF-α and inhibit glucose transport in adipose cells (adipocytes) [While the exact pathogenic mechanisms involved in the association between POP and the MetS have not been established with certainty, results from a number of studies indicate that insulin resistance and changes in gene regulation caused by POP play the major role. POP can increase secretion of tumor necrosis factor alpha (TNF-α), which plays an essential role in development of insulin resistance. The insulin resistance-inducing effect of TNF-α is established –41. 2,3,pocytes) ,42. TCDDpocytes) . The undpocytes) . TNF-α spocytes) . TNF-α apocytes) .et al. (2006) investigated the NF-κB:GLUT4 ratio in the exposed veterans, as compared to the veterans without history of exposure, and found that this ratio is significantly correlated with serum dioxin levels [et al. (1999) found elevated levels of insulin in non-diabetic veterans [The role of the TNF-α → NF-κB → GLUT4 pathway in POP-induced glucose intolerance was also demonstrated in a study of United States Air Force veterans of the Vietnam War who were involved with spraying the Agent Orange herbicide that was contaminated with TCDD. Fujiyoshi n levels . In anotveterans . High inAlthough investigating MetS-associated hospitalization in relation to demographic characteristics was not a goal of our study, these results may be worth a brief discussion. Prevalence of the MetS is known to increase with age ,47. Our Our study is not free from limitations. Exposure status information was available only on a zip code-level (group level), which is a very crude measure of exposure. Group-level measurements are a common limitation intrinsic to ecological and semi-ecological studies. Therefore, the results of such studies cannot be extrapolated to individual level because of ecological fallacy problem ,50. It sIt can be reasonably argued that crude measurement of exposure might have produced a dilution effect, thus weakening the measure of association (underestimation of RR) due to non-differential misclassification. That means that if exposure status was measured more accurately, the true RR obtained in such a study would have been even higher, indicating stronger association between exposure and outcome. Since exposure and outcome status were estimated independently from each other, using independent sources of data, differential misclassification is unlikely in our study.5.In conclusion, our findings contribute to the body of evidence supporting the hypothesis of POP constituting an environmental risk factor for the MetS. Further studies of POP exposure and the MetS with individual level measurements of exposure and outcome, including measures of insulin resistance, are warranted. Given the involuntary nature of environmental residential exposure to POP that may affect a substantial portion of population, it is imperative to develop and implement respective public health and environmental health policy interventions aimed at reducing and eliminating POP exposure.


We assessed the effect of a web-based, self-directed sensitivity training on MSM for HCWs ; this was most pronounced in those with clinical or administrative roles and in those from governmental health providers. Compared to baseline, homophobic attitudes had decreased significantly three months after training, particularly among HCWs with high homophobia scores at baseline, and there was some evidence of correlation between improvements in knowledge and reduction in homophobic sentiment.Seventy-four HCWs  from 49 health facilities were trained, of whom 71 (96%) completed all measures. At baseline, few HCWs reported any prior training on MSM anal sexual practices, and most HCWs had limited knowledge of MSM sexual health needs. Homophobic attitudes were most pronounced among HCWs who were male, under 30 years of age, and working in clinical roles or government facilities. Three months after training, more HCWs had adequate knowledge compared to baseline (49% vs. 13%, McNemar's test Scaling up MSM sensitivity training for African HCWs is likely to be a timely, effective and practical means to improve relevant sexual health knowledge and reduce personal homophobic sentiment among HCWs involved in HIV prevention, testing and care in sub-Saharan Africa. Sub-Saharan Africa has a very high burden of HIV-1 infection, of which a substantial proportion occurs among populations reporting high-risk sexual behaviour such as transactional sex and anal intercourse . As a reSocietal discrimination on the grounds of sexual orientation has been reported frequently among African MSM, taking the form of sexual, physical and verbal assault –7, and acalled to action to reduce stigma and discrimination, provide integrated services for mental health concerns and substance use, screen MSM routinely for HIV and sexually transmitted infections (STIs) and ensure training for all personnel in clinical settings [Health worker training, social mobilization and community engagement were prioritized as structural interventions in a recent consultation on priority areas for MSM HIV prevention research involving 69 participants from 17 African countries . HCWs hasettings . As yet,settings .Since 2005, biomedical research has been ongoing with both HIV-1 negative MSM and MSM living with HIV in coastal Kenya . To dateInternet-based learning (e-learning) as a cognitive tool has increasingly been used in health professions in resource-constrained low- and middle-income countries . E-learn“MSM: An introductory guide for health workers in Africa” is a paper-based HCW sensitization training first developed in 2010. The content of training was validated and revised through a programme of extensive classroom use in South Africa, and following expert review . The papThe objectives of this study were (1) to assess the feasibility of facilitated self-directed learning of MSM health issues in coastal Kenya and (2) to evaluate the effect of the training intervention upon HCW knowledge and attitudes.Seventy-four HCWs involved in HIV prevention, treatment and care services in coastal Kenya were recruited to participate in the study. We mapped 54 ART-providing governmental and nongovernmental health providers in four districts in coastal Kenya . An average of two staff representatives from each health-providing facility were invited to the training intervention, including clinicians and counsellors as well as clinic administrators.Four “district AIDS/STD coordinators” (DASCOs) working within the study districts were trained to lead the MSM sensitization training during a 2-day “training-of-trainers” course similar to the training proper. An additional day was used to prepare focus-group topic guides and organizational matters. The study procedures were approved by the ethical review board at the Kenya Medical Research Institute, and all participants provided written informed consent for impact evaluation. HCWs received Ksh2000 (approximately US$24.00) to cover travel expenses and lodging.http://www.marps-africa.org) and group discussions. The curriculum consisted of the following modules of study: (1) MSM and HIV in sub-Saharan Africa; (2) Stigma; (3) Identity, coming out and disclosure; (4) Anal sex and common sexual practices; (5) HIV and sexually transmitted infections; (6) Mental health, anxiety, depression and substance abuse; (7) Condom and lubricant use; and (8) Risk reduction counselling. Modules were designed to be self-completed in 1–2 hours each, including multiple-choice questions  at the end of each module. A score of 71% correct was required to advance to the next module, and upon successful completion of all eight modules, participants were sent a link to download their course certificate. A post-course evaluation asked for opinions and suggestions for course improvements, using both closed and open-ended questions.The training consisted of two consecutive days and included eight modules which were taken in four sessions . Each session was followed by a group discussion. Each group size comprised 18–19 participants. DASCOs were supported by four members of the research team  and two members of a local LGBTI  organization. HCWs were introduced to the sensitivity training on MSM health issues and learned that the training consisted of computer-assisted learning , details of working practice  and specific experience working with HIV prevention, treatment and care with the most at-risk populations (MARPs) in Africa. To assess baseline levels of knowledge, participants conducted a pre-course 24-item multiple-choice assessment covering key learning outcomes across the course material, and they completed a 25-item Homophobia Scale ,Good (17–22 questions correctly answered) and Excellent (>22 questions correctly answered). When 17 or more questions were correctly answered, the immediate post-training knowledge was considered adequate.I have damaged property of gay persons, such as “keying” their cars, was replaced with Homosexuality should be treated as an illness/Homosexuality can be cured (question 17); I would feel comfortable with having a gay roommate was replaced with Homosexuality is un-African/is something brought by foreigners (question 18); and I have rocky relationships with people that I suspect are gay was replaced with Gay men have the same rights to public/tax-funded services as straight men (question 25). Responses to items 1, 2, 4, 5, 6, 9, 12, 13, 14, 15, 17, 18, 19, 21, 23 and 24 were reverse coded . The total HS score (HSS) was the sum of all item scores, with 25 subtracted from the total. The range is between 0 and 100, with an HSS of 0 being the least homophobic and 100 being the most homophobic.The HS, which was developed and standardized among college students in the United States by Wright et al. , was adaAnalysis was conducted using Stata 11.0 . Binary and categorical characteristics of study participants, established at baseline, were compared using chi-square tests. Although both knowledge and HS scores before and after training approximated to Gaussian distributions, differences between paired measures were non-normal, and thus unadjusted nonparametric methods were used for analysis. Median differences between pre- and post-training knowledge and homophobia score are reported with an interquartile range (IQR). A Wilcoxon signed rank test for matched pairs was applied to test the statistical significance of differences between pre- and post-training scores. Mann–Whitney and exact McNemar's tests were used to test differences in scores and binary measures, respectively, by HCW characteristics. Spearman's rank was used to assess correlation between pre- and post-training scores, and knowledge and HS scores at both points. Multivariate linear regression models of pre- and post-training score outcomes were explored, but they yielded no additional insight beyond bivariate analysis.2p<0.01).Seventy-four HCWs were recruited to participate in the training programme, and their characteristics are shown in Most HCWs (73% (54/74)) required 1 hour or less to complete each module, whilst 11% (8/74) required 2 hours or more per module. While 68% (50/74) considered the course duration to be “just right,” 23% (17/74) said that it was too short, and 9% (7/74) that it was too long. Overall, by the end of training, 60/74 (81%) HCWs reported feeling empowered to discuss MSM behaviour and anal sex in their professional work.All participants said they would recommend the course to others. Open-ended suggestions for course improvements are presented in p=0.293).2p<0.001 vs. pre-training). At three months after the course, 35 (49%) of the 71 HCWs reassessed had retained “adequate” knowledge compared to 9/71 (13%) at pre-training (exact McNemar's χ2p<0.001). This represented a significant increase in the median assessment score of 12% (IQR 4–21%) between baseline and three-month knowledge assessments (Wilcoxon signed test for matched pairs p<0.001). Significant sustained improvements in knowledge were apparent for all HCW age groups and genders, those with clinical or administrative roles and those from governmental health providers.At the end of training, 70/74 (95%) HCWs had adequate course knowledge (exact McNemar's χp< 0.001), indicating that improvements in knowledge tended to be highest among HCWs with lower pre-training knowledge. There were no significant differences in the degree of knowledge gain by the gender or age group of HCWs; however, participants in counselling roles achieved significantly lower gains in sustained knowledge than other HCWs .Pre-training and three-month post-training scores were negatively correlated , and HCWs working in government facilities had significantly higher HS scores than HCWs in NGOs (p=0.037).p<0.001), reflecting the tendency for HCWs with high pre-training HS scores to exhibit greater decreases in this measure as a result of training  compared to their baseline HS score; in four (5.6%), HS scores were unchanged; and in 14.1% (10/71), HS scores were higher after training than before. Overall, the median decrease in individual HS score after training was 8 points (IQR 2–15), which was statistically significant. These findings did not change in a sensitivity analysis omitting the three HS questions that were culturally adapted (data not shown). Individual pre-training and post-training HS scores were negatively correlated .Male HCWs and those working in clinical roles and in governmental institutions recorded the most pronounced reductions in HS score subsequent to training, although differences in median reduction comparing HCWs’ gender, age group, staff role and institution were not statistically significant. More modest declines in HS score were apparent for counsellors (median reduction after training: 4 points) and staff of NGOs (median reduction after training: 0 points); however, it is notable that these groups reported relatively low HS scores prior to training. Collectively, there was some evidence for correlation between scale of increase in individual knowledge and scale of decline in HS score, and this was of borderline statistical significance  requires Kenyan HCWs to document the number and category of MSM using HIV services.Whilst targeted services may well be necessary for subpopulations of MSM, such as male sex workers, they are unlikely to replace the need for MSM-specific clinical care among general health services. MSM-specific programmes have aroused considerable social antipathy in Kenya to date  and may The combination of self-directed, modular computer-based learning supplemented by group discussions facilitated by trainers identified from within the existing workforce may offer a relatively sustainable and mobile model for episodic health professional training in this context. The learning content of this course is freely available as a web resource, yet reliable access to internet services remains elusive and expensive in most parts of the country. Even where it is available, the narrative reflections by participants who undertook this training emphasize the importance of the sanction provided by facilitated group discussions to share and explore personal and professional issues arising from the training content that may well be lost in self-directed learning .The brief training programme described here resulted in significant improvements in knowledge of MSM sexual health issues pertinent to day-to-day prevention and clinical practice, and it was sustained by most trainees until at least three months after training. Increase in knowledge was accompanied by a reduction in negative attitudes toward MSM over the same period. Encouragingly, the positive effect of training upon knowledge and personal attitudes toward MSM was strongest among HCWs who had poor levels of knowledge and/or more extreme negative attitudes toward MSM prior to training. That positive changes were most marked among HCWs in clinical roles within governmental settings, which represent the backbone of Kenyan HIV services, is cause for particular optimism. Studies to date of perceived barriers to healthcare access identified by MSM in Kenya ,30 and eThis study has a number of limitations. The HS, which was originally developed and validated among college students in the United States , requireWe report qualitative narratives among HCWs returning to their workplace after training but finding little support for new perspectives amongst (untrained) colleagues . In a reFinally, the ultimate goals of improving knowledge of MSM sexual health needs and reducing prejudicial attitudes toward MSM in healthcare settings are to enhance the accessibility of population-based, public health services to MSM themselves. Although surely a prerequisite to accessible HIV prevention, treatment and care for MSM, the extent to which changes in the attitudes and practices of healthcare providers are reflected in the perceived and practical accessibility and acceptability of services to MSM themselves is unknown. Further study will be required to establish the effect of this brief intervention on long-term attitudes and professional practices towards MSM, and what practical contribution such strategies might make to addressing unmet HIV-related needs among MSM.In summary, we developed, implemented and evaluated a brief training intervention addressing knowledge and attitudes toward MSM and their sexual health needs in Kenya. The training, which combined self-directed and facilitated group learning, increased health worker knowledge and reduced homophobic attitudes up to three months after training. Scaling up such interventions offers a straightforward response to the immediate need to support HCWs in offering accessible and informed services to address the largely sexual health needs among MSM in Kenya.


Metabolic syndrome (MetS) is associated with an increased risk of the development of atherosclerotic cardiovascular disease (CVD). Interleukin-18 (IL-18), which is a pleiotropic proinflammatory cytokine with important regulatory functions in the innate immune response system, plays a crucial role in vascular pathologies. IL-18 is also a predictor of cardiovascular death in patients with CVD and is involved in atherosclerotic plaque destabilization.In order to determine if circulating levels of IL-18 can serve as a specific biomarker for distinguishing MetS patients from pre-MetS subjects, we studied 78 patients with visceral fat deposition and 14 age-matched control subjects. Increased levels of IL-18 were observed more frequently in patients with MetS than in pre-MetS subjects and were positively associated with waist circumference. Serum levels of IL-18 were significantly reduced by a change in weight caused by lifestyle modifications. There was a significant interaction between waist circumference and serum IL-18 concentration. Weight loss of at least 5% of the body weight caused by lifestyle modification decreased IL-18 circulating levels relative to the reduction in waist circumference and blood pressure, suggesting that this degree of weight loss benefits the cardiovascular system.IL-18 may be a useful biomarker of the clinical manifestations of MetS and for the management of the risk factors of CVD. Obesity and the related metabolic syndrome (MetS) are major public health problems  that areInterleukin-18 (IL-18), which is a pleiotropic proinflammatory cytokine with important regulatory functions in the innate immune response system, plays a crucial role in vascular pathologies. IL-18 is also known as a predictor of cardiovascular death in CVD patients and is involved in atherosclerotic plaque destabilization. A growing body of evidence suggests that IL-18 levels may be closely related to MetS and its consequences -13. IncrWe hypothesized that circulating levels of IL-18 may enhance atherosclerosis-prone conditions in patients with MetS. In order to examine this hypothesis, we studied the circulating levels of IL-18 in MetS patients and in subjects with pre-MetS conditions. Furthermore, the circulating levels of IL-18 were examined in MetS patients before and after lifestyle modifications that resulted in weight loss.In order to determine whether the circulating levels of IL-18 could be a specific biomarker to distinguish MetS patients from subjects with pre-MetS conditions, we studied 42 patients with MetS or pre-MetS and 14 control subjects . There were 28 patients diagnosed as having MetS , and the remaining 14 patients were designated as pre-MetS , which was defined as the subjects having only 1 component of the MetS criteria proposed by the Japanese Society of Internal Medicine. The baseline characteristics of the subjects are shown in Tables As shown in Table Increased levels of glycated hemoglobin (HbA1c), CRP, and IL-18 were observed in MetS patients Table . DecreasP < 0.01), and these levels were positively associated with fasting insulinemia (P < 0.05). Interestingly, serum levels of IL-18 were slightly, but significantly, correlated with the waist circumference in patients with MetS and pre-MetS conditions .We hypothesized that body weight loss from lifestyle modification would improve systemic inflammation in patients with MetS. Serum IL-18 levels were measured in 57 patients with MetS  before and after they lost at least 5% of their initial weight by lifestyle modification. As shown in Table P < 0.01).Among all of the subjects undergoing lifestyle modifications, 89% achieved significant reductions in weight, waist circumference, HOMA-IR, and blood pressure after the lifestyle modification was maintained for an average of 22.6 5.6) weeks. As shown in Table .6 weeks.P < 0.01), and the levels significantly correlated with the change in weight (P = 0.046). Lifestyle modification-induced weight loss of 5% body weight significantly improved the levels of circulating biomarkers that are related to metabolic and vascular inflammation (Table Serum levels of IL-18 were significantly reduced with weight loss ( P = 0.04. Lifestyon Table .P = 0.015). A 5% weight loss from lifestyle modification decreased the circulating levels of IL-18 in relation to the reduction in waist circumference, serum CRP levels, and blood pressure, suggesting that this degree of weight loss resulted in cardiovascular benefits.The reduction in IL-18 concentration correlated with increases in adiponectin  who consulted for medical care in the Metabolic Syndrome Clinic, Department of Cardioangiology, Kitasato University Hospital. All subjects provided informed consent before participating in this study, and anonymity was maintained by tracing the patients through their clinical history number.The project was approved by the Scientific and Ethical Committee of the Kitasato University School of Medicine, Japan. BMI was calculated as weight divided by height squared. Systolic and diastolic blood pressures were measured after a rest of at least 15 min with a sphygmomanometer while subjects were in a sitting position. HOMA-IR, which was used as a measure of insulin resistance, was calculated as fasting plasma insulin (μU/mL) × glucose (mg/dL)/405 . All subMetabolic scores were calculated using MetS components according to the MetS criteria proposed by the Japanese Society of Internal Medicine . The scoAfter an overnight fast, blood serum samples were collected by venipuncture from 78 patients with abdominal obesity and from 14 healthy donors. The age range of the healthy donors matched that of the patients. For the IL-18 biomarker study examining the differences in IL-18 levels between MetS patients and subjects with pre-MetS conditions, 36 patients out of the 64 patients with MetS were excluded because they were taking metabolic-mimetic agents .Biochemical markers such as triglycerides, LDL cholesterol, HDL cholesterol, insulin, plasma glucose, HbA1c, uric acid, gamma-glutamyl transpeptidase (γ-GTP), CRP, and BNP were measured.In order to examine the effects of lifestyle modification in the IL-18 biomarker study, 64 patients with MetS were recruited to lose weight in order to improve their cardiovascular risk factors. We studied 57 patients with MetS  who successfully lost at least 5% of their initial weight through lifestyle modification. All subjects were instructed to maintain a standard mild energy-restricted diet and to engage in walking for at least 30 min a day, 5 days a week. Serum IL-18 and adiponectin levels were measured in these subjects before and after lifestyle modification, which was maintained for 8-34 weeks.Circulating levels of human IL-18 and adiponectin were determined by an enzyme-linked immunosorbent assay (ELISA) using the human IL-18 ELISA Kit  and the CircuLex™ human adiponectin ELISA Kit , respectively. Samples were processed according to the manufacturer's instructions ,50.t-test or Mann-Whitney U-tests, where appropriate. Differences in the proportions of variables were determined by a chi-squared analysis. In order to evaluate the relationship between IL-18 and selected variables, we calculated Spearman correlation coefficients between the circulating levels of fasting IL-18 and the following variables: (1) conventional risk factors for CVD , and history of coronary artery disease; (2) measures of adiposity and insulin resistance ; and (3) metabolic risk scores  and MetS-related co-morbid conditions .Continuous data are summarized as either mean (SD) or median and quartiles, and categorical data are expressed as percentages. The data were compared by an unpaired P-values of < 0.05 were considered significant.In order to evaluate the association of IL-18 with MetS, we constructed multivariable logistic regression models in order to assess whether the circulating IL-18 levels were independently associated with MetS. We calculated the odds ratio for the presence of MetS in each IL-18 level quartile, and the participants with IL-18 levels in the lowest IL-18 quartile were considered as the reference group. Adjustments were performed for age and sex and for age, sex, and β-blocker use. Two-sided The authors declare that they have no competing interests.MY-T participated in the design of the study and performed the statistical analysis. TT conceived of the study, and participated in its design and coordination and helped to draft the manuscript. Other authors participated in enrolling patients in the study and discussion. All authors read and approved the final manuscript.


Mobile HIV screening may facilitate early HIV diagnosis. Our objective was to examine the cost-effectiveness of adding a mobile screening unit to current medical facility-based HIV testing in Cape Town, South Africa.per capita Gross Domestic Product (GDP) . We projected that, with medical facility-based testing, the discounted (undiscounted) HIV-infected population life expectancy was 132.2 (197.7) months; this increased to 140.7 (211.7) months with the addition of the mobile unit. The ICER for the mobile unit was $2,400/year of life saved (YLS). Results were most sensitive to the previously undiagnosed HIV prevalence, linkage to care rates, and frequency of HIV testing at medical facilities.We used the Cost Effectiveness of Preventing AIDS Complications International (CEPAC-I) computer simulation model to evaluate two HIV screening strategies in Cape Town: 1) medical facility-based testing (the current standard of care) and 2) addition of a mobile HIV-testing unit intervention in the same community. Baseline input parameters were derived from a Cape Town-based mobile unit that tested 18,870 individuals over 2 years: prevalence of previously undiagnosed HIV (6.6%), mean CD4 count at diagnosis , CD4 count-dependent linkage to care rates , mobile unit intervention cost . We conducted extensive sensitivity analyses to evaluate input uncertainty. Model outcomes included site of HIV diagnosis, life expectancy, medical costs, and the incremental cost-effectiveness ratio (ICER) of the intervention compared to medical facility-based testing. We considered the intervention to be “very cost-effective” when the ICER was less than South Africa's annual The addition of mobile HIV screening to current testing programs can improve survival and be very cost-effective in South Africa and other resource-limited settings, and should be a priority. An estimated 6 million people in South Africa are living with HIV/AIDS, and 300,000 die annually of their disease HIV counseling and testing on mobile units has begun to be implemented throughout sub-Saharan Africa and has been targeted by the South African government as a strategy to introduce HCT services directly into communities medical facility-based HIV testing, in which outpatients undergo diagnostic testing via a primary health care clinic's pre-existing HCT program; or 2) the mobile unit intervention as well as medical facility-based testing (hereafter referred to as the mobile unit intervention), the addition of a one-time offer of an HIV test and POC CD4 count via a mobile testing unit for those HIV-infected. Both strategies are simulated in the same community; undiagnosed HIV-infected patients can also link to clinical care following presentation with an AIDS-defining opportunistic infection (OI). Cohort characteristics are derived from a study of patients tested in a mobile unit in Cape Town, South Africa from March 2010–September 2011 We use a widely-published HIV disease simulation model, the Cost-Effectiveness of Preventing AIDS Complications International (CEPAC-I) model, to evaluate the clinical and economic value of adding a mobile HIV testing unit, including point-of-care (POC) CD4 count testing, to current medical facility-based HIV testing in Cape Town per capita Gross Domestic Product (GDP) , and “cost-effective” if less than three times the South Africa annual per capita GDP HIV-related costs are assessed from a modified societal perspective (excluding patient travel time and lost wages) and are reported in 2012 US$. We report all outcomes used to inform resource allocation decisions on a present-value basis using a 3% discount rate We use the CEPAC-I model, a validated state transition computer model, to simulate natural history, screening, engagement in care, clinical management and costs of HIV disease. We use both the CEPAC-I Screening and Disease Models , which hWe simulate HIV screening at the population level and account for both HIV-infected and HIV-negative individuals. HIV testing and linkage to care occur in one of three ways: 1) following presentation with an AIDS-defining OI ; 2) via Individuals in both screening strategies experience the same HIV disease progression, response to treatment, engagement with care  and mortality. In the Disease Model, all HIV-infected individuals undergo monthly transitions between health states that depend on CD4 count, HIV RNA, and incidence/history of acute OI infection. Declines in CD4 count increase the risk of OIs and HIV-related mortality. After diagnosis and linkage to care, patients receive guideline-concordant care with CD4 count and HIV RNA monitoring, and are eligible for prophylaxis and ART To characterize the simulated cohort for both the medical facility and mobile unit testing strategies, we use population and clinical data, as well as health care utilization and cost data from a mobile testing unit deployed in Cape Town, South Africa In the medical facility-based strategy, the average HIV testing frequency is once every 5.7 years among HIV-negative individuals and once every 4.0 years among HIV-infected individuals  [18]. FoIn the mobile unit intervention strategy, all individuals regardless of HIV status are offered a one-time rapid HIV test. The probability of accepting an HIV test, accepting a CD4 count, and linking to care are varied independently, and are not 100%. HIV test acceptance probabilities are derived from the likelihood of HIV test acceptance among individuals who enter the Cape Town mobile unit . Reactive results are confirmed by a second HIV rapid test. At the same visit, individuals with a positive HIV test are offered a POC CD4 count. Successful linkage to care is defined as a clinic visit within 3 months of mobile unit testing. Linkage to care is stratified by CD4 count and accounts for poorer linkage at higher CD4 counts. For individuals with an observed CD4 count ≤350/µL, linkage to care probabilities are stratified by observed CD4 count (49%–58%). For individuals with an observed CD4 count ≥350/µL, linkage to care probabilities are stratified by sex  because of significant differences noted by gender in the Cape Town mobile unit. In both males and females, linkage to care generally increases as observed CD4 count decreases, consistent with the literature ART is initiated upon diagnosis of a severe AIDS-defining OI or TB, regardless of CD4 count, or with a CD4 count ≤350/µL The cost to receive an HIV test in the medical facility-based program is $9.30 per negative result and $13.90 per positive result; these costs include the initial and confirmatory HIV tests, staff salaries and space in a voluntary counseling and testing site Costs are calculated based on the Cape Town mobile unit's expenditure and utilization  [26]. WeOn average, the Cape Town mobile unit operated 3 days per week, testing approximately 40 people per day, with fluctuations due to national holidays, weather, staffing, training and other activities. For the remaining 2 days of the work week, the mobile unit was deployed for an incentivized HIV testing program which is not included as part of this study. Assuming the mobile unit operates 5 days per week, at the capacity recorded from March 2010–March 2011, we determine that it could have tested 18,870 people over a 2-year intervention period All patients, regardless of where they were diagnosed, are subject to the same medical costs of HIV-related care. These costs include inpatient and outpatient services, laboratory tests, and ART regimens, for those eligible to be on ART.In sensitivity analyses, we vary multiple input parameters within plausible ranges to evaluate the impact on the cost-effectiveness results for mobile unit testing. Initially, we vary each major model parameter at a time, including the prevalence of undiagnosed HIV, mean age of testers, medical facility-based program average per-person HIV testing frequency, mobile unit HIV test and POC CD4 count acceptance probabilities, linkage to care probabilities, intervention cost, HIV test and POC CD4 count test costs, as well as the probability of LTFU and return to care. We then evaluate the interaction of the most influential parameters in multi-way sensitivity analyses.Based on widely-cited guidelines for the conduct of budget impact analysis The study was approved by the University of Cape Town Health Sciences Faculty Ethics Committee (SA), and the Partners Health Care Human Research Committee (Protocol 2010P002636), Boston, Massachusetts, USA.In the medical facility-based strategy, the discounted life expectancy was 249.9 months  in the overall population and 132.2 months  in the HIV-infected population . The addThe addition of the mobile unit intervention to current medical facility-based testing increased the discounted average per-person lifetime costs from $3,970 to $4,070, yielding an incremental cost-effectiveness ratio of $2,400/year of life saved (YLS) for the mobile unit intervention compared to medical facility-based testing.In the medical facility-based testing strategy, 21% of HIV-infected people died before linking to care and 26% were linked to care after they developed a severe AIDS-defining OI (including TB) . The remFive-year survival among the HIV-infected population in the medical facility-based testing strategy was 69% compared to 73% in the mobile unit intervention strategy . The mobIn one-way sensitivity analyses, we varied major input parameters independently within plausible ranges in both strategies see . The ICEper capita South African GDP unless the prevalence of undiagnosed HIV was below 0.5% and linkage to care was less than 70%. Even with annual HIV testing provided by the medical facility-based program, the addition of the mobile unit intervention to medical facility-based testing had an ICER <3× GDP unless the prevalence of undiagnosed HIV was below 1%.We used multi-way sensitivity analyses to evaluate the interplay of the most important input parameters identified in one-way sensitivity analyses: prevalence of undiagnosed HIV, linkage to care, and the medical facility-based testing program per-person testing frequency . With meFor the 18,870 people projected to be evaluated by the mobile unit over 2 years , the total undiscounted HIV-related costs over the 2 years increased by $900,000 (from $1.5 million to $2.4 million), an increase of 67% compared to the facility-based testing strategy . Roughlyper capita GDP of South Africa, which we considered to be very cost-effective The successes of mobile units in sub-Saharan Africa demonstrate the potential for major benefit with mobile unit HIV testing and integrated staging by POC CD4 in South Africa and other resource-limited settings The mobile unit intervention remained very cost-effective in nearly all sensitivity analyses and was particularly robust to changes in the mobile unit costs including intervention start-up, operation, maintenance, and laboratory tests. The mobile unit remained very cost-effective even if targeted to the lowest provincial prevalence of undiagnosed HIV in South Africa . These results indicate that the potential benefits from mobile screening persist even with major improvements in medical facility-based testing, such as might be realized with antenatal clinic testing, and underscore how a combination of extensive medical facility-based with mobile unit HIV testing offers the potential for immense benefit in South Africa and elsewhere.Mobile testing units facilitate HIV diagnosis prior to advanced disease per capita South African GDP .Given the cost-effectiveness of the Cape Town mobile HIV testing unit, as well as the successes of a variety of programs within sub-Saharan Africa, mobile HIV testing units are clinically effective and feasible options in many resource-limited settings As the population in any one area is saturated with HIV testing, we anticipate a decrease in prevalence of undiagnosed HIV making marginal returns non-linear. A benefit, however, of mobile testing is that the yield (e.g. new HIV diagnoses) can be assessed in real time (e.g. on a monthly basis). As a result, regular deployment and re-deployment of a number of mobile units can be targeted to areas where the prevalence of new HIV diagnoses remains sufficiently high enough to make testing cost-effective, and mobile units could be moved to other areas with higher prevalence or eventually phased out of service.Is mobile unit HIV testing economically feasible in sub-Saharan Africa? Mobile units have been deployed in urban and rural settings in multiple countries, including South Africa, Nigeria, Zimbabwe, Tanzania and Uganda There are several limitations to this analysis. First, we assumed that, with the exception of the mean CD4 count at the time of HIV diagnosis, the cohort characteristics and HIV disease progression were the same for the medical facility-based testing and the mobile unit intervention groups. This may, however, miss social and economic differences between the cohorts that could influence patients' ability to access and stay in care. Second, we based the prevalence of undiagnosed HIV for both the medical facility and mobile unit on data collected by the mobile unit. Though this may have underestimated the undiagnosed HIV prevalence at the medical facility as people often go to medical facilities when sick, we performed sensitivity analyses to explore uncertainty in this parameter. Third, we used data from multiple studies to define the cohort and in some cases were limited by availability of data. Although we stratified some input parameters by sex, we did not have sex-specific data on undiagnosed HIV prevalence, viral load, response to treatment, or AIDS-, OI- and toxicity-related mortality. While we anticipate that having sex-specific parameters might alter the results slightly, there is no reason to believe they would change policy conclusions. Fourth, we did not consider new HIV cases that would develop during the intervention, so only prevalent cases were considered over the 2-year period with the focus on one-time testing. Fifth, we did not account for the monetary benefits of HIV transmissions averted as a result of increased ART coverage with mobile HIV screening, nor did we quantify the benefits of transmissions likely averted due to risk reduction associated with HIV screening The now well-established benefits of timely access to ART necessitate a strong commitment to effectively diagnose HIV-infected individuals and ensure that they link to and remain in care. In South Africa, the National Department of Health and the National Health Treasury have expressed the political and financial will to create more opportunities for frequent, regular HIV testing Appendix S1The technical appendix includes further details on the CEPAC model, model inputs and sources, and sensitivity analyses.(DOCX)Click here for additional data file.Figure S1One-way sensitivity analyses on the addition of mobile unit HIV testing to medical facility-based testing. This tornado diagram summarizes the results of multiple 1-way sensitivity analyses on the incremental cost-effectiveness of the addition of mobile unit HIV testing to medical facility-based testing in Cape Town, South Africa. The horizontal bars represent the incremental cost-effectiveness ratio (ICER) range as a result of variations in each single model parameter. The solid vertical line indicates the base case ICER . The dashed vertical line indicates the South Africa per capita gross domestic product . YLS: years of life saved; POC: point of care. ; SOC: standard of care.(TIFF)Click here for additional data file.Figure S2Two-way sensitivity analyses on mobile unit test acceptance and linkage to care. This diagram shows the incremental cost-effectiveness of the addition of mobile unit HIV testing to medical facility-based testing under conditions of varied mobile unit test acceptance and linkage to care. Linkage to care is varied on the vertical axis and test acceptance is on the horizontal axis.(TIF)Click here for additional data file.


Constructivist learning theory contends that we construct knowledge by experience and that environmental context influences learning. To explore this principle, we examined the cognitive process relational complexity (RC), defined as the number of visual dimensions considered during problem solving on a matrix reasoning task and a well-documented measure of mature reasoning capacity. We sought to determine how the visual environment influences RC by examining the influence of color and visual contrast on RC in a neuroimaging task. To specify the contributions of sensory demand and relational integration to reasoning, our participants performed a non-verbal matrix task comprised of color, no-color line, or black-white visual contrast conditions parametrically varied by complexity . The use of matrix reasoning is ecologically valid for its psychometric relevance and for its potential to link the processing of psychophysically specific visual properties with various levels of RC during reasoning. The role of these elements is important because matrix tests assess intellectual aptitude based on these seemingly context-less exercises. This experiment is a first step toward examining the psychophysical underpinnings of performance on these types of problems. The importance of this is increased in light of recent evidence that intelligence can be linked to visual discrimination. We submit three main findings. First, color and black-white visual contrast (BWVC) add demand at a basic sensory level, but contributions from color and from BWVC are dissociable in cortex such that color engages a “reasoning heuristic” and BWVC engages a “sensory heuristic.” Second, color supports contextual sense-making by boosting salience resulting in faster problem solving. Lastly, when visual complexity reaches 2-relations, color and visual contrast relinquish salience to other dimensions of problem solving. The human brain collates, integrates, and binds basic sensory inputs that result in our capacities to think, reason, and perform at high levels  contribute additional sensory load to relational reasoning resulting in extended behavioral reaction times, and (2) that those differences are observable and dissociable in the visual and prefrontal cortices. Thus, hypothesizing that real-world visual properties play a supportive role in relational reasoning, our open question was under which levels of complexity and to what extent do properties of the visual environment influence the RC system in the frontal cortex to enable successful decision making and choice?To our knowledge, this study is the first to document the convergence of color perception and RC during a self-paced reasoning task, representing one example of how perceptual processes enable higher level cognition in the context of a task format commonly used to assess problem solving skill and general intellectual capacity.SD = 7.5). All volunteers were right-handed with a mean score of 86.48% on the Edinburgh Handedness Inventory  is a self-paced, event-related design incorporating 3 × 3 matrix reasoning problems missing the bottom right figure in No-color line (NC) Figure , Color , and BWVPrior to their functional magnetic resonance imaging (fMRI) session, participants performed a practice test with samples of trials at different complexity levels to avoid a learning confound. In the MRI, they had a brief practice with the button box, using their right hand index, middle, ring, and small fingers to press one of four buttons corresponding to their choice. Problem trials were presented from a computer and back-projected through a wave-guide onto a screen located in the back of the MRI scanner bore. Visual images were viewed from a mirror mounted on the head coil above the participant's head.During the fMRI experiment, participants were alerted to the beginning of each trial by a visual cue  followed by a matrix problem Figure . WorkingTR = 3000 ms, TE = 30 ms, flip angle = 70°, FOV = 192 × 192 mm2, 64 × 64 voxels). Functional images covered the whole brain, collected in 870 volumes per participant over the course of 3 runs of 14.5 min each. Each volume consisted of 48 interleaved slices with 0.2 mm gap and thickness of 3.0 mm. The first three volumes were discarded in order to account for signal stabilization. The start of each run was manually synchronized with the scanner.Structural and functional MRI data were acquired using a 3.0-tesla Siemens Allegra head-only scanner  located at the Krasnow Institute for Advanced Study at George Mason University. A CP TX/R head coil single-channel was used during data collection and head motion was restricted with memory foam inserts to secure and cushion the head within the head coil. Functional runs were acquired using a standard BOLD (blood-oxygenation-level-dependent) gradient-echo echo-planner imaging (EPI) pulse sequence  and additional structural images with following parameters: spin-echo axial-oblique T1-weighted structural scans of the whole brain  = 200 ms, echo time (TE) = 3.6 ms, field of view (FOV) = 192 × 192 mm2; slice thickness = 3 mm, flip angle = 75°; matrix size = 205 × 256).Structural images were collected as high-resolution T1-weighted images using a MPRAGE (magnetization-prepared rapid-acquisition gradient-echo) sequence with following parameters: (Image reconstruction was performed offline. The conversion of raw data completed using MRIconvert (University of Oregon Lewis Center for Neuroimaging). Data processing and analysis were carried out using the Statistical Parametric Mapping software package SPM5 . Pre-processing included slice-timing, reorientation, and realignment using INRIAlign .A general linear model (GLM) was applied to the time course of activations to estimate condition effects at each voxel. In the first-level single subject analysis, the response function was modeled as an RT-based boxcar function  analysis was used to address our a priori hypothesis that the RLPFC would support RC within a stimulus context that included real world visual properties. To explore the signal more precisely for Color 1- and 2-relational problems , we performed both structural and functional ROI analysis. Structural ROIs were created according areas identified from previous studies that used similar paradigms  than for Color (BA18/19), illustrating a neural efficiency for how color primes cognitive performance.When controlling for stimulus complexity, the rostrolateral prefrontal cortex (RLPFC), the suite of areas that specifically support relational integration during reasoning, is preferentially engaged during Color RC. This is observed in a double dissociation between visual and frontal areas that engage for BWVC (working memory) and for Color (RC) Table .Second, our main physiological findings can be understood in two points:Participants demonstrated a mean level accuracy of 95.7% (standard error = 0.44%). The analysis of data for systematic decrease in accuracy or systematic increase in RT from the beginning to the end of each run did not indicate a fatigue effect. Accuracy percentage and mean RT per condition were computed for each participant and averaged for each condition and complexity level Figure . As the post-hoc analysis of RTs showed a significant increase corresponding to the increase in the level of complexity within each condition   and for Color 0-relational trails . However, the same comparison at 1-RC level showed a significant decrease in RT for Color problems  and no significant change for BWVC. Within 2-RC level, no significant changes were assessed between No-Color and Color or between No-Color and BWVC. A comparison between Color 0-relation and BWVC 0-relation trials did not show any significant change.A One-Way ANOVA with repeated measures and a ion [F8, 4 = 129.9http://en.wikipedia.org/wiki/Brodmann_area). Brodmann Areas are then listed in the Tables p < 0.05.Neuroimaging results include 34 participants and report activations identified through voxel-based analysis using an RT-convolved HRF. We analyzed the results for the effect of complexity level 0, 1, 2) and for the effect of each condition . For complexity level, contrasts were analyzed within and between conditions by evaluating 1-relational vs. 0-RC and 2-relational vs. 1-RC. For the effect of each condition, contrasts were performed between conditions at equal level of complexity. All neuroanatomical results are reported in Tables  and for First, we predicted that color and BWVC would contribute additional sensory demand during relational reasoning. This was confirmed in our behavioral results demonstrating that RTs increase as a function of complexity within BWVC and Color conditions Table . By contp < 0.05) threshold including: right BA8, left BA6, bilateral BA10. In posterior sensory areas, significance appears in dorsal stream visual areas bilateral BA7, and BA18 in the ventral stream  analysis across different complexity levels in areas found to be significant Tables , 5. A siFinally, to further examine the pattern of response, Finite input response (FIR) curves were modeled for Color 1-relational and 2-relational conditions in each statistically significant ROI. The hemodynamic response modeled in FIR had similar time course trends in right middle frontal gyrus (BA10), left middle frontal gyrus (BA6), and precuneus (BA7) for each of Color 1- and 2-RC conditions. Also, FIR models in left and right superior parietal lobules (BA 7) and inferior occipital gyrus (BA18) showed a similar trend in their response pattern for Color 2-RC problems.This experiment examined the influence of specific properties of the visual environment (color and BWVC) on neural systems of RC during non-verbal matrix reasoning. Our aim was to better understand how consilient real-world visual perception influences decision-making by dissociating contributions of sensory demand from higher level cognitive processing during relational reasoning. To examine this phenomenon, we used a self-paced, event-related neuroimaging design, incorporating matrix reasoning problems similar to the RPM Raven,  construcThis experiment accomplished three aims. First, we were able to dissociate the ways in which color and BWVC contribute additional sensory demand during relational reasoning. When color and BWVC are each compared with the control condition, no color line matrices, neural systems in visual cortex become more localized as problem complexity increases. Black and white visual contrast systems are more salient in the 0-relational condition. Color systems are more salient in the 1-relational condition. This is an artificial comparison because our visual world is comprised of each of these properties. In practical terms, color and visual contrast assist with our perceptual assessment of safety (demarcating boundaries), to our sense of place and capacity to navigate, and to our aesthetic sensibilities for appreciating art, design, architecture, other visual arts and aspects of our daily environments. Importantly, we demonstrate how each property contributes to sensory perception that supports sense-making during problem solving.Second, elaborating on this discussion, color plays a specific priming role demonstrated by its influence on behavioral RT when an initial change is introduced (1-relation) such that RT significantly varies from both the control and the BWVC condition. This boost in salience is reflected as less RT needed to problem solve at this level.Lastly, as predicted, this experiment provides direct evidence for the functional connection of the RC system in the RLPFC with ventral and dorsal streams in visual cortex known to support color perception, visual-spatial processing, and navigation. This connection is demonstrated by areas of visual and parietal cortex that are activated with frontal lobe areas that specifically support relational reasoning. As described in the forthcoming paragraphs, we performed an additional region of interest analysis (ROI) to show that the time course and shape of the BOLD response in these sensory areas of the brain  correspond to BOLD responses assessed in the relational reasoning areas of the brain  Figures , 6. The Our fMRI results provide evidence that BWVC enlists a “sensory heuristic” during relational reasoning indicated by the multiple areas of the visual cortex listed in Table P < 0.001 (Z > 3.09) threshold. Within the no color 2-relational vs. 1-relational contrast, activations were localized in BA6, BA8, and BA10 in the RLPFC. Within the BWVC condition, the 2-relational vs.1-relational comparison, activations appear in right BA9, left postcentral gyrus, right BA40, right BA18/19 and left BA18, but are only significant at an uncorrected statistical threshold (p < 0.001). This validates previous studies of RC and situates this experiment to extend our knowledge of the role of RC in reasoning and problem solving.Consistent with previous studies , we demonstrate a double-dissociation between the areas of the prefrontal and visual-parietal cortices for color and BWVC Table . During The neural correlates that accompany increases in RT suggest that color is a visual property that contributes to cognitive demand in RC. The between condition contrast, color vs. no color, illustrated that the addition of color to RC trials requires additional processing from parietal and ventral occipital areas to process visual information. This is important in light of the evidence that perception of context is engaged early in decision-making and relies on the dorsal stream as a primary pathway for information transfer between the visual and frontal cortices  would be influenced by color? We assessed this by examining the Color 2 vs. Color 1 within relational contrast. Our results provide evidence of the recruitment of systems beyond those in the prefrontal cortex that appear necessary for color relational reasoning, extending the known system in RLPFC to include relevant superior parietal and inferior occipital areas Table . The neuColor 2-RC problems in our task recruited bilateral BA7. Activations in BA7 have been previously associated with working memory, setting attention priority, potential movement coding . This is an important distinction, the distinction between perception of color as a general property in the visual environment and color as an object property of a stimulus in the neuroimaging environment. Early experiments exploring aspects of complexity within several different visual properties determined that features of symmetry and context reduce the psychological perception of complexity Chipman, , a potenper se. Our choice to use the color blue as a starting point came from the fact that it is a color most people can perceive, including those with color-blindness (Deeb, It is worth noting a potential limitation but important feature of our study. We did not vary color as a dimension of RC ss Deeb, . Had we ss Deeb, . Also, bss Deeb, .In total, this experiment yields results that discern rules for how the brain employs different and specific reasoning and sensory heuristics for processing color and visual contrast to show how these features are assimilated during problem solving. By demonstrating this within the context of matrix reasoning, we have preserved the relevance of neuroimaging to inform our understanding of the assessment of human intelligence and to influence the design and optimization of formal and informal physical and virtual learning environments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Barriers to HIV treatment among injection drug users (IDU) are a major public health concern. However, there remain few long-term studies investigating key demographic and behavioral factors - and gender differences in particular - that may pose barriers to antiretroviral therapy (ART), especially in settings with universal healthcare. We evaluated access and adherence to ART in a long-term cohort of HIV-positive IDU in a setting where medical care and antiretroviral therapy are provided free of charge through a universal healthcare system.We evaluated baseline antiretroviral use and subsequent adherence to ART among a Canadian cohort of HIV-positive IDU. We used generalized estimating equation logistic regression to evaluate factors associated with 95% adherence to antiretroviral therapy estimated based on prescription refill compliance.p = 0.042). After adjusting for clinical characteristics as well as drug use patterns measured longitudinally throughout follow-up, female gender was independently associated with a lower likelihood of being 95% adherent to ART .Between May 1996 and April 2008, 545 IDU participants were followed for a median of 23.8 months (Inter-quartile range: 8.5 - 91.6), among whom 341 (63%) were male and 204 (37%) were female. Within the six-month period prior to the baseline interview, 133 (39%) men and 62 (30%) women were on ART (Despite universal access to free HIV treatment and medical care, female IDU were less likely to access and adhere to antiretroviral therapy, a finding that was independent of drug use and clinical characteristics. These data suggest that interventions to improve access to HIV treatment among IDU must be tailored to address unique barriers to antiretroviral therapy faced by female IDU. During the past decade, there have been significant advances in the treatment of HIV disease with the advent of antiretroviral therapy (ART) . ART hasAlthough newer, simplified ART regimens have enhanced treatment adherence , specifiBritish Columbia, Canada has a universally accessible, publicly funded healthcare system without user fees or other financial barriers to medical services, including all HIV/AIDS care. This allows for investigation of HIV-related outcomes without the potentially confounding effect of financial barriers to medical care and HIV treatment that may be present in other settings. Therefore, we conducted the present study to investigate factors associated with adherence to ART among a Canadian cohort of HIV-positive IDU and specifically examined if gender differences in adherence to ART existed in this context.The AIDS Care Cohort to evaluate Exposure to Survival Services (ACCESS) is a prospective observational cohort of HIV-seropositive injection drug users (IDU) in Vancouver, Canada. The cohort has been described in detail previously ,16,17, aAs previously described ,16,17, tIn the present study, we included all participants who were recruited and completed at least one interview between May 1996 and April 2008, and excluded only those where clinical data were unavailable. As indicated above, the primary independent variable of interest was gender and, to compare rates of ART use at baseline, we evaluated access to ART at the time of recruitment into the cohort by examining the proportion of participants who had been on ART in the 6-month period prior to the baseline interview. Since we had long-term prospective data, we then assessed the longitudinal pattern of ART exposure by examining ART adherence during the six-month period preceding each semi-annual follow-up visit throughout follow-up. As in previous studies using this validated and confidential pharmacy dispensation data ,18, we m10copies/mL) and CD4 cell count (per 100 cells/mm3), using the closest measure within one year of the baseline.We considered explanatory variables potentially associated with the dependent variable including: gender ; age (<24 yrs vs. ≥24 yrs); ethnicity ; daily injection heroin use (yes vs. no); daily cocaine injection (yes vs. no); daily crack cocaine smoking (yes vs. no); current methadone use (yes vs. no); any treatment use (yes vs. no); education (less than high school vs. other); employment ; and unstable housing (yes vs. no). Age was defined as a dichotomous variable according to the World Health Organization's definition of a 'young person', using the upper age limit of 24 as the cut-off . All dicWe began by comparing the rate of ART use at baseline. We then examined univariate associations between the explanatory variables and ART adherence throughout follow-up. Because serial measures for each variable were available for each subject, we used generalised estimating equations (GEE) for the analysis of correlated data. This approach allows for the identification of factors associated with the outcome over the entire study period. Data from every participant follow-up visit were considered in this analysis. Missing data were addressed through the GEE estimating mechanism which uses all available pairs method to encompass the missing data from dropouts or intermittent missing. All non-missing pairs of data are used in the estimators of the working correlation parameters. Standard errors were calculated using an exchangeable correlation structure, adjusted by multiple observations for each individual. GEE models have routinely been used to analyse datasets containing repeated measures, including longitudinal IDU cohorts ,21.a priori defined model building protocol whereby we included all explanatory variables with a univariate p-value < 0.05. We also ran the models with interaction terms for key independent variables in order to better compare rates of adherence between men and women. We conducted additional sub-analyses adjusting for key clinical or demographic characteristics and for the year of the baseline interview to account for advances in antiretroviral therapy during the study period. All statistical procedures were performed using SAS version 9.1 . All p-values are two-side.Following examination of the univariate results we fit a multivariate GEE logistic regression model using an p = 0.042). There were 1186 (26.6%) periods where individuals were adherent out of 4460 total observations. Overall, 81 (19%) out of 422 males and 83 (29%) out of 287 females were excluded from the analysis as a result of missing baseline CD4 count or viral load.Between May 1996 and April 2008, 545 participants were eligible for the present study, and there was a median of 23.8 months (Inter-quartile range: 8.5 - 91.6) of prospective follow-up. Of these study participants, 341 (63%) were male and 204 (37%) were female. The characteristics of the study population stratified by gender are shown in Table p < 0.001), and accessing any addiction treatment  were associated with being 95% adherent to ART, whereas higher baseline viral load , education , daily heroin injection , daily cocaine injection , and female gender  were negatively associated with 95% adherence. Table The results of the univariate GEE logistic regression demonstrated that methadone use  was independently associated with 95% ART adherence. Higher baseline viral load , age less than 24 years , daily heroin injection , daily cocaine injection ; education ; and, female gender  were independently and negatively associated with 95% adherence to ART. There were no statistical interactions observed in the multivariate GEE analysis. Table Multivariate GEE regression demonstrated that methadone use .We conducted several sub-analyses of behavioural and clinical variables. A sub-analysis was conducted defining drug use as a 6-level categorical variable using 'daily injecting' as the reference level showed results that were consistent with analyses defining drug use as a dichotomous variable (data available from the corresponding author). Sub-analyses adjusting for year of the baseline interview, to account for advances in antiretroviral therapy during the study period, were consistent with the primary analysis (data available from the corresponding author). Additionally, a sub-analysis of time-updated clinical variables to determine if clinical outcomes affected adherence or drug using behaviours demonstrated that, although cocaine use was no longer significant, the gender effect remained ; and through an Avant-Garde Award (No. 1DP1DA026182-01) from the National Institute of Drug Abuse, at the US National Institutes of Health. He also received funding from Merck, Gilead and ViiV to support research into Treatment as Prevention.EW, CT, and MJM drafted the manuscript. RZ performed the statistical analysis.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/86/prepub