Current cervical cancer screening guidelines recommend a Pap test every 3 years for women age 21–65 years, or for women 30–65 years who want to lengthen the screening interval, a combination of Pap test and high-risk human papilloma virus testing (co-testing) every 5 years. Little population-based data are available on human papilloma virus test utilization and human papilloma virus infection rates. The objective of this study was to examine the patient-level, cervical cancer screening, and area-level factors associated with human papilloma virus testing and infection among a diverse sample of uninsured and underinsured women enrolled in the New Jersey Cancer Early Education and Detection (NJCEED) Program.We used data for a sample of 50,510 uninsured/underinsured women, age ≥ 29 years, who screened for cervical cancer through NJCEED between January 1, 2009 and December 31, 2015. Multivariable logistic regression models were used to estimate associations between ever having a human papilloma virus test or a positive test result, and individual-  and area-level covariates , and number of screening visits.Only 26.6%  of the sample had at least one human papilloma virus test. Among women who underwent testing, 13.3% (1792) tested positive for human papilloma virus. Most women who were positive for human papilloma virus (99.4%) had their first test as a co-test. Human papilloma virus test utilization and infection were significantly associated with age, race/ethnicity, birthplace (country), and residential area-level poverty. Rates of human papilloma virus testing and infection also differed significantly across counties in the state of New Jersey.These findings suggest that despite access to no-cost cervical cancer screening for eligible women, human papilloma virus test utilization was relatively low among diverse, uninsured and underinsured women in New Jersey, and test utilization and infection were associated with individual-level and area-level factors. Approximately 80 million people are currently infected and 14 million people are newly infected annually with human papillomavirus (HPV) in the United States (US) . AlthougIn 2004, HR-HPV DNA tests were approved by the US Food and Drug Administration (FDA) for use concurrently with cytology (co-testing) as a primary cervical cancer screening test for women age ≥ 30 years –7. Most Little population-based data exist on the utilization of and barriers/facilitators to HPV DNA testing in the US, particularly for minority and medically underserved women with a higher risk of developing cervical cancer. The limited population-based studies on HPV co-test utilization have generally demonstrated low utilization across various population subgroups (approximately ≤20% testing rates) –12; howeAlthough rates of cervical cancer incidence in New Jersey has been declining dramatically from 1990  to 2014  at approximately 2.7% per year  [The NJCEED Program is housed within the New Jersey Department of Health (NJDOH) and is funded primarily by the NJDOH and the Centers for Disease Control and Prevention (CDC) to provide screening, diagnostic, and treatment services to eligible individuals for breast, cervical, prostate and colorectal cancers. Eligibility for receipt of NJCEED program services, include low-income  and lack of adequate health insurance (uninsured or underinsured). NJCEED has been providing breast and cervical cancer outreach, education, early detection, screening and follow-up to eligible individuals in New Jersey since 1993 with initial funding through the CDC’s Preventive Health and Health Services Block Grant. Statewide coverage, with services being provided in all 21 counties, began on September 1, 1997. Currently, NJCEED program services are provided through 21 contracted lead agencies, with at least one lead agency in each county. The lead agencies work directly with providers within their county to provide services to eligible program participants. Between January 1, 2000 and December 31, 2015, a total of 116,313 unique and eligible women, age 21 to 64 years, received cervical cancer screening services through NJCEED. Among these, 55,827 were enrolled during the study period of interest . We further restricted our study sample to women age ≥ 29 years, who had available data on race/ethnicity and residential zip code , which includes data on invasive cancer cases ascertained through annual data linkages with the New Jersey State Cancer Registry (NJSCR). CaST allows the user to track patients through the length of the study and collect information on screening and diagnostic procedures done for breast and cervical cancer (NBCCDEP) and colorectal cancer (CRCCP). Individual-level data for this study were obtained from the Minimum Data Elements (MDEs) of the CaST based on inclusion criteria described above. All identifiers were removed prior to providing the limited data set to study investigators for analysis. Use of the data and all related study activities were approved by the New Jersey Department of Health, Rutgers Biomedical Health Sciences Institutional Review Board, and the Scientific Review Board of Rutgers Cancer Institute of New Jersey.Age at enrollment was defined as the age at the earliest cervical cancer screening visit during the study period (2009–2015). Race/ethnicity and place of birth were based on patient self-report and stored in CaST. A total of 166 unique countries of birth were reported among participants, which were collapsed into 7 categories: (1) USA, (2) Central and South America, (3) Caribbean, (4) Africa, (5) Asia and the Middle East, (6) Europe, Russia, Australia and Oceania, and (7) Other Countries. In terms of cervical cancer screening variables, number of cervical cancer screening visits was defined as the total number of unique visits to NJCEED providers for any cervical cancer screening procedures at any time during the study period ; and number of HR-HPV DNA tests was defined as the total number of unique visits where an HPV test was performed during the study period. HR-HPV DNA test results were only available as positive result or negative result. Specific HR-HPV DNA types  for positive results were not available. Age at first Pap test and HPV test were defined as study participant’s age at the earliest Pap test or HPV test, respectively, reported during the study period.Residential zip code tabulation area (ZCTA) for each study participant was linked to the 2010–2014 American Community Survey (ACS) 5-year ZCTA-level estimates collected by the US Census Bureau. The following ACS estimates were obtained to derive area-level sociodemographic factors: (1) poverty status in the past 12 months; (2) race/ethnicity; (3) health insurance coverage type; and (4) language spoken at home. Proportions of participants falling into quintiles  of each of the above sociodemographic factors (based on ZCTA population estimate distributions within the NJCEED population in our study cohort) were estimated. The proportion of minority residents per ZCTA was calculated as the difference of 1 minus the proportion of non-Hispanic White (NHW) residents. Age-adjusted cervical cancer incidence and mortality rates by New Jersey county were obtained from the National Cancer Institute (NCI) State Cancer Profiles [P < 0.05 level. County level rates of HR-HPV DNA testing, positive results for HR-HPV DNA test, and invasive cervical cancer incidence and mortality were also examined. Data were analyzed using SAS v9.4  and STATA v14 .Our primary outcomes of interest are having at least one HR-HPV DNA test and having ever received a positive HR-HPV DNA test. Descriptive statistics were used to summarize the individual sociodemographic and screening characteristics. Chi-square tests were used to compare these variables by HR-HPV DNA test receipt and receipt of a positive HR-HPV DNA test result. We conducted bivariate logistic regression models to examine the relationship between individual-level covariates , number of screening visits, and area-level measures with our primary outcomes. Full models were based on significant relationships in the unadjusted models. Tests for multicollinearity were also conducted, with negative results. We ran multivariate models adjusting for year, which was significantly associated with HR-HPV DNA testing rates but not HR-HPV positivity. Upon inclusion of year in the full model, the associations remained mostly consistent. Study participants with missing demographic or zip codes were excluded from analysis. We estimated odds ratios (OR) and 95% confidence intervals (CI) using robust standard errors and defined significance as Among the 50,510 women included in the analysis, 26.6%  had at least one HR-HPV DNA test through NJCEED over the 6-year study period from 2009 to 2015. Among those who ever had a HR-HPV DNA test, 13.3% (1792) had received a positive test result. Characteristics of the analytic cohort overall, as well as among those ever tested for HR-HPV and among those who ever received a positive HR-HPV test result are shown in Table P < 0.0001). Compared to women who were never tested for HR-HPV test, those who were tested were more likely to be younger at enrollment into NJCEED, younger at their first Pap and HR-HPV tests, to have more cervical cancer screening visits, to have more Pap tests, and were less likely to be NHW and US-born . Almost all women who had a HR-HPV DNA test through NJCEED had their first test as a co-test with Pap test (99.8%) and the first test result was positive among 12.7% of these women. Like women who had a HR-HPV test, participants who tested positive for HR-HPV were also more likely to be younger at enrollment in NJCEED, younger at their first Pap and HR-HPV DNA tests, to have more cervical cancer screening visits, and to have more Pap tests compared to those who never tested positive for HR-HPV . However, unlike the women who were tested, HR-HPV positive women were more likely to be NHW and US-born. Almost all women who had ever received a positive HR-HPV test results (99.4%) had their first test as a co-test with a Pap test and for 95.5% of them their first HR-HPV test was positive.In terms of cervical cancer screening, most women had only one screening visit (59.1%) and had only one Pap test (70.6%) over 6 years. Additionally, they had their first NJCEED Pap test an older age . In terms of HR-HPV testing, 73.4% of the sample were never tested. HR-HPV DNA testing rates among women age ≥ 29 years increased from 7.4% in 2009 to 35.4% in 2015  , while several other counties had HR-HPV infection rates that were 2- to 3-fold higher than the sample average . Figure In our analysis of HR-HPV DNA testing and HR-HPV infection by New Jersey county of residence, we found that the rates of HR-HPV DNA testing among residents of eight counties were significantly higher than the 26.6% overall rate of testing in the study sample  among this sample of eligible women. We also observed wide variation in HR-HPV DNA test utilization and HR-HPV infection by individual-level  and area-level characteristics (poverty), as well as by county of residence. Of note, while non-Hispanic Black and Hispanic patients had higher odds of HR-HPV DNA test use, they were significantly less likely to be HR-HPV-infected compared to NHWs.Few studies have examined the rates of HR-HPV DNA testing in cervical cancer prevention –12, but Similar to our current study, another study  examininWe observed wide variation in HR-HPV DNA test utilization and HR-HPV infection by individual-level and area-level characteristics. After controlling for covariates, NHBs, APIs, and Hispanics (compared to NHWs) and residents of ZCTAs with larger proportions of residents living below FPL (quintile 5 vs. quintile 1) had higher odds of ever being tested for HR-HPV. The higher odds of HR-HPV DNA testing among NHBs, Hispanics, and women living in high poverty areas were particularly interesting given that there are striking disparities in the cervical cancer burden in these groups, despite widespread reductions in incidence and mortality. Minority, uninsured and low socioeconomic status (SES) women tend to be screened less often and this lower screening tends to result in increased cervical cancer morbidity and mortality –21. ThesWe also found that the odds of HPV testing decreases as age increases, a finding which has been reported by other investigators . AdditioWe found that among women who ever had a HPV DNA test, 13.3% of them had at least one positive test, indicating infection with one or more HR-HPV types. Our finding that NHB and Hispanics had lower odds of ever having a positive HPV test, although having higher odds of ever having a HPV DNA test, was unexpected, given existing data that show higher rates of HPV infection among low SES, minority women, which has been linked to their disproportionate burden of cervical cancer , 35–38. The observed variation in HR-HPV DNA test utilization by New Jersey county of residence identified several counties with rates higher than the average of the study sample, which indicates that NJCEED providers covering these areas are doing relatively well at providing guideline cervical cancer screening in medically underserved populations in the state. However, we also identified several counties that had HPV screening rates that were quite low. While we did not examine provider characteristics in the current study, we hypothesize that they are important to understanding variation in cervical cancer screening and HPV DNA testing across counties and by NJCEED providers. Prior studies have shown that variation exists in providers’ adherence to screening guidelines and specifically in recommendations for HPV testing , 39–42.The strengths of this study include its large sample size of diverse, uninsured and underinsured women who sought cervical cancer screening services through NJCEED during the study period. Availability of cervical cancer screening history through large administrative data for several years was also a strength. An additional strength is that our findings will contribute to the dearth of population-based data related to HR-HPV test utilization and HR-HPV infection in a diverse sample of women. There were also some limitations of this study that should be considered in the interpretation of our findings. First, the use of administrative data from the NJCEED Program limited the availability of additional covariates of interest, including specific individual-level SES measures and more detailed information on sexual behaviors and other risk factors for HR-HPV infection and HPV vaccination status among study participants. Additionally, we cannot confirm that women included in this study did not seek cervical cancer screening outside of NJCEED during the study period. We also saw a high proportion of women who were only screened once within the program, based on their encrypted participant ID number. While some of these women may have repeat screenings within NJCEED across our study years, they may have been assigned a different participant ID due to screening at a different location or other reasons. Another limitation was that we don’t know if any of the NJCEED providers or sites targeted screening for high-risk groups (and the variation among providers), which could explain some of the observed differences in county-level testing rates. We also don’t know if NJCEED is reaching all of the women who are eligible for the program and if all of these women are seeking cervical cancer screening services through the program, if providers are meeting their catchment area targets for the NJCEED Program, and how much funding they have to meet the needs of the women in their catchment area. Thus, generalizability of these data to women not eligible for NJCEED services is not possible. And finally, lack of data on specific HR-HPV genotypes prevented us from examining the type specific prevalence of HR-HPV by subgroup of women, which would really enhance our assessment of cervical cancer risk in this population.This is one of the few studies to examine utilization of HR-HPV DNA testing and HR-HPV DNA infection among a sample of medically underserved women who are at high-risk for cervical cancer. These study findings provide insight to individual, area-level factors associated with testing and positive testing results as well as geographic variation across the state in HR-HPV DNA testing, which can inform further exploration of barriers to low utilization of HR-HPV DNA testing, particularly among high risk populations and to develop strategies to increase the utilization rates of this important cervical cancer screening test.


To determine the transplacental pharmacokinetics of the HIV integrase inhibitor dolutegravir.Maternal-to-fetal transfer across the term human placenta was investigated with the ex-vivo dually perfused cotyledon model, in 5 closed-circuit, recirculating experiments. Dolutegravir was added to a maternal perfusate containing antipyrine, a marker to validate the cotyledon’s viability, and 2 g/liter of human albumin.After 3h of recirculating perfusion, the mean (± SD) DTG concentrations in the maternal and in the fetal compartments were respectively 2450 ± 286 ng/mL and 715 ± 369 ng/mL, with a fetal-to-maternal ratio of 34% ± 18% and a clearance index (in comparison with antipyrine transfer) of 79% ± 23%. The mean cotyledon accumulation index was 153% ± 25%.Fetal transplacental exposure to dolutegravir was considerable as well as accumulation in placental tissue. Whether this may lead to risks for the exposed fetus requires more investigation. Sustained suppression of plasma viral load is the key to preventing vertical transmission from HIV-infected women to their children. In the prospective multicenter French Perinatal Cohort (ANRS-EPF), no mother-to-child-transmission of HIV-1 occurred among nearly 3000 women receiving antiretroviral therapy (ART) before conception and throughout the pregnancy, with a plasma VL <50 copies/mL at delivery , but theDolutegravir is an HIV-1 integrase strand transfer inhibitor (INSTI) which is increasingly used and recommended by the World Health Organization for a universal first-line of ART, in combination with nucleoside reverse transcriptase inhibitors or rilpivirine . WhereasBecause clinical trials are ethically difficult in pregnant women, other preclinical approaches are required in order to obtain data on the exposure of the fetus to medications given to the mother. The ex vivo human cotyledon is an accepted model used to study and interpret placental transfer of drugs –18.The purpose of this study was to investigate the placental transfer of dolutegravir in the ex vivo human perfused cotyledon.Placentas were collected after uncomplicated pregnancy with full-term delivery  in mothers who were seronegative for HIV and hepatitis B and C viruses and had taken no medication other than vitamin supplements and perimedullar analgesia. They were collected in the maternity ward of Louis Mourier Hospital, Assistance Publique-Hôpitaux de Paris, Colombes, France, and were rapidly perfused on site. Women who donated a placenta gave their written informed consent. The research project was approved by the Institutional Review Board .Dolutegravir was obtained by crushing commercial tablets (each film-coated tablet of Tivicay contains 50 mg of dolutegravir sodium), after which the powder was weighed, solubilized in dimethyl sulfoxide  and prepared in separate tubes, in order to contain 3 mg of dolutegravir for each experiment. The target was to obtain, after dilution in the maternal compartment, concentrations for the active drug as observed in adults taking dolutegravir 50 mg once daily, where Cmax is on the order of 3000 ng/mL . ExperimRecirculating/closed perfusions were performed. The perfusate which leaked from the intervillous space into the perfusion chamber was recirculated into the maternal reservoir.Samples were collected every 5 min over 180 minutes to determine the concentrations of dolutegravir and antipyrine in the fetal and maternal compartments and stored at -20°C until analysis. Dolutegravir concentrations in maternal and fetal samples were determined using ultraperformance liquid chromatography coupled with tandem mass spectrometry  . AnalyteRetention of dolutegravir in the placental cotyledon tissue was also determined, and expressed as an accumulation index (AI), defined by the ratio of the concentration of the study drug in the cotyledon (ng/mg) multiplied by the tissue density (1.04 g/mL) divided by the mean maternal drug concentration of the relevant experiment.Five procedures were validated  for wild-type HIV of 64 ng/ml for dolutegravir [After 3h of perfusion , the meategravir .At the end of the recirculating/closed perfusion, the mean (SD) cotyledon accumulation index was 153% ± 25%.Dolutegravir crossed the placenta, with a fetal-to-maternal ratio of 34% after 3 hours of perfusion in the closed-circuit model, at maternal concentrations in line with the in vivo average plasma concentrations obtained following once-daily oral administration 50 mg dolutegravir in HIV-infected patients. In that experiments, fetal concentrations exceeded the in vitro protein-binding adjusted IC90 values (64 ng/mL) . We obseThe moderate molecular weight and lipophilic nature of dolutegravir  , favor iWhen performing ex vivo placental perfusions without albumin, the transfer parameters for protein-binded molecules are modified. Schalkwijk et al  reportedThe placental transfer of dolutegravir seems to be higher as compared with the other integrase inhibitors, raltegravir and elvitegravir. For raltegravir, Vinot et al  found anThe ex vivo cotyledon model is a non-invasive approach which has no ethical restrictions in order to study placental transfer of medications which have not yet been tested in pregnant women. It allows for evaluation in standardized and highly controlled conditions. Recirculating studies imitate physiological conditions and can be used to evaluate transplacental transfer and metabolism, while in open perfusions drug clearance can be calculated. Both of these approaches showed concordant results, and in addition the placental transfer appeared to be independent of the initial maternal concentration. Furthermore, placental accumulation, which is rarely analyzed in placental perfusion studies, is of interest to interpret placental metabolism and transport.However, there are well-known limitations to the ex vivo perfusion model , 15, 34.Placental transfer of dolutegravir is sufficient to entail in utero exposure of the fetus. Knowledge about placental transfer is important for clinical decision-making. High transfer to the fetus can protect it from perinatal transmission of HIV, acting as pre-exposure prophylaxis (PreP) , and dolS1 TableDTG: dolutegravir. ATP: antipyrine. CF: Concentration in fetal circulation. CM: Concentration in maternal circulation.(XLSX)Click here for additional data file.


Antidepressants are among the most-prescribed class of drugs in the world and though weight gain is a common outcome of antidepressant treatment, that effect is not well understood. We employed an animal model comprised of 2 weeks of chronic restraint stress with antidepressant treatment, followed by diet-induced obesity. We showed that short-term antidepressant treatment had long-lasting effects, not only leading to weight gain, but also enhancing trabecular and cortical bone features in rats; therefore, weight gain in this model was different from that of the classic diet-induced obesity. Late in the post-restraint recovery period, antidepressant-treated animals were significantly heavier and had better bone features than saline-treated controls, when assessed in the distal femoral metaphysis. The propensity to gain weight might have influenced the rate of catch-up growth and bone allometry, as heavier animals treated with fluoxetine also had enhanced bone features when compared to non-stressed animals. Therefore, short-term antidepressant treatment ameliorated the long-term effects of stress on body growth and bone. Growth and bone structural features were associated with leptin levels, and the interaction between leptin levels and antidepressant was significant for bone mineral content, suggesting that short-term antidepressants in the context of long-term diet-induced obesity modified the role of leptin in bone formation. To our knowledge this is the first study reporting that short-term antidepressant treatment has long-lasting effects in restoring the effects of chronic stress in body weight and bone formation. Our findings may be relevant to the understanding and treatment of osteoporosis, a condition of increasing prevalence due to the aging population. According to the data from the World Health Organization (WHO), MDD has become the second most prevalent cause of illness-induced disability, affecting 350 million people worldwide3. Concomitantly, obesity is a debilitating epidemic, affecting 36.5% of US adults4. Currently, given the high prevalence of obesity and mood disorders, it is conceivable that nearly 25% of the cases of obesity may be attributable to the association with MDD5. Cross-sectional and longitudinal studies have been conducted in order to understand the casual relationship between MDD and obesity8. Both disorders have in common the dysregulation of the hypothalamic–pituitary–adrenal axis, which is persistently activated during chronic stress9.Major depressive disorder (MDD) and obesity are both common heterogeneous disorders of complex etiology, and pronounced public health impact4. Weight gain is a common outcome of antidepressant treatment. The interplay between MDD, obesity, and antidepressant-induced weight gain is complex. Though acute selective serotonin reuptake inhibitor (SSRI) treatment leads to weight loss, chronic SSRI treatment may lead to weight gain12. The discontinuation rate for antidepressant treatment is high within 2 months of treatment initiation, ranging from 70% for fluvoxamine, to 45% for fluoxetine (FX) and 40% for sertraline;15 thus, the lifetime prevalence of antidepressant exposure is very high. Based on the model that we have described previously16, here we combined 2-weeks of recurrent restraint stress and antidepressant treatment, followed by long-term diet-induced obesity; and referred it as the stress-antidepressant and diet-induced obesity (SADIO) model.In the USA, antidepressant drugs were the second most prescribed class of drugs in 2011–201417 included increased bone length, weight and structural changes. Furthermore, there was a signficant association between leptin and bone mineral content (BMC) in the SADIO model, which was not present in animals not exposed to antidepressants.We hypothesized that increased body weight related to antidepressant treatment in the SADIO model had different pathophysiological mechanisms from those of the diet-induced obesity model. In the SADIO model described in this paper, we show that previously described body changes in the post-stress acclimation and recovery periodMale Sprague-Dawley rats  were housed one per cage at 24 °C and with a 12-h light/dark schedule (07:00 h to 19:00 h) in a stress-free environment. All the animal experimental procedures conducted were approved under protocol number J.MB.50.10, Animal Experimentation Ethics Committee, The Australian National University, Australia.N = 30). The NR group did not receive CRS or intraperitoneally (i.p.) injections, but received the same dietary schedule as the CRS groups. For overview of the experimental protocol see supplementary Figure SN = 13, 10 mg/kg i.p.; Sigma-Aldrich, St Louis, MO, USA], fluoxetine  or saline solution  immediately prior to the CRS procedure. Imipramine was prepared in 24.5 mg/mL in 0.9% sodium chloride solution by mild vortexing and pH was adjusted to 7.4. The solution was further diluted and the final concentration of the imipramine was 4.9 mg/mL in 0.9% sodium chloride solution. Fluoxetine hydrochloride was prepared 5.6 mg/mL in 0.9% sodium chloride due to its low solubility and pH was adjusted to 7.4. The solution was further diluted to final concentration of 4.9 mg/mL in 0.9% sodium chloride solution. Solutions were filtered with a 0.22-μm filter . After the CRS period, all groups of animals received a high-fat diet  to induce obesity from day 19 to 296 (276 days). Researcher could not be blinded to the experimental groups as daily i.p. injection of specific treatment group was necessary from days 5 to 19. We did not conduct power analysis prior to this study as there was no study using our animal model for 2 weeks of CRS followed by induced obesity from day 19 to 296. Body weight and food intake were measured daily during the CRS period and twice weekly thereafter. Due to the fact that rodents, such as Sprague-Dawley rats, display variable weight gain when fed a high-fat diet, animals were classified into subgroups of animals that gain significant weight (obesity prone) or not (obesity resistant)18. Therefore, we conducted analyses to understand whether the ability to gain weight during diet-induced obesity had significant effects in our model. Within each group, rats in the upper 50% of body weights were classified as the obesity-prone subgroup based on their body weight at the end of the experiment, and animals in the lower 50% were classified as the obesity-resistant subgroup.Animals were randomly allocated into four groups: three received chronic restraint-stress (CRS) and one group did not receive CRS . Total distance (TD), center distance (CD), and center distance to total distance (CD/TD) ratio were obtained and used as an index of anxiety16. Groups were CRS treated with saline, N = 11; FX, CRS treated with fluxotine, N = 14, and IM, CRS treated with imipramine, N = 11. Their means were subsequently averaged along the 10 sessions. Elevated plus maze (EPM): During the post-stress acclimation period (day 257), EPM test was performed to measure the level of anxiety-like behavior. EPM was elevated from the floor with two open arms and two closed arms (50 cm × 13 cm). Rats were placed in the middle of the maze facing a closed arm; trials were conducted for 5 min/session. The number of entries into the open arms was counted and the percentage of time spent in the open arms (ratio of open arms time/closed arms time) was calculated. The groups were CS, N = 11; FX, N = 12; IM, N = 11. Researchers were blinded while conducting behavioral tests.Open-field tests and elevated plus maze were conducted during the post-stress acclimation period , equipped with a camera tracking system . N = 4; FX, N = 5; IM, N = 4; NR, N = 10.Body composition analysis of subsets of obesity-prone (upper 30%) and obesity-resistant (lower 30%) animals were obtained under anesthesia using the GE Lunar PIXImus equipment  and the Lunar imaging software (ver.1.46) according to standard procedures . Distal femora were scanned submerged in 70% ethanol at a resolution of 21.3 μm. Transverse plane images were obtained from reconstructed images using the DataViewer software (Skyscan), and distal metaphyseal volume and mid-diaphyseal cortical geometry were generated by a binarized image program  , leptin , triglyceride , total cholesterol , free fatty acid , vanillylamandelic acid , and pituitary GH , following the manufacturers’ protocols  days 20–60 and (B) day 60 onward. Analyses performed in R v3.4.1  using the nlme package  or non-parametric test of Kruskal-Wallis when the variances were not equal among treatment groups, and respectively followed by Tukey’s or Dunn’s post hoc test, using the GraphPad Prism 5.0 software  , bone mirenal density (BMD), % fat, body length, body weight) with four biochemical measures  were tested using linear regressions (see P < 0.001) than the non-stressed reference (NR) group . Furthermore, the FX group became heavier than the NR group .Immediately post CRS, groups treated with FX, imipramine (IM) or vehicle (CS) had significantly lower body weight , we conducted behavioral tests on animals that had undergone CRS. The FX group was significantly less anxious than the CS group, based on the open field test measure of CD/TD ratio, and on the open/closed arm ratio on the elevated plus maze test  were significantly lighter than obesity-prone FX and obesity-prone NR animals  had significantly lower lean mass and fat mass in comparison to the obesity-prone NR subgroup, and lower lean mass than obesity-prone FX animals  had lower BMD and BMC in the DXA scan, in comparison to the obesity-prone NR and obesity-prone FX subgroups .In all the groups, we measured plasma lipid profile , followed by chronic intake of high-fat diet (>9 months) on body composition and bone morphology. In this study, we evaluated body weight recovery patterns, body composition, bone features, biochemical and gene expression measurements during the post-stress acclimation period, which is a recovery phase in which the animal is free from the stressor exposure17. In agreement with previous literature, 2 weeks of CRS induced significant weight loss in our animals  group did not attain full body weight recovery in comparison to the NR group, while both antidepressant-treated groups had much better body weight recovery. The ability of the SADIO model to recover from CRS-induced weight loss during the post-stress acclimation period could be referred to as a phenomenon of “catch-up growth”, which is growth that occurs after a period of growth-inhibiting conditions23. Our results suggest that CRS and antidepressant treatment resulted in a period of greater rate of catch-up growth during the post-stress acclimation period. The IM group made full body weight recovery and the FX group had an exacerbated body weight recovery, recovery weights of the FX groups were significantly larger than that of the CS group. These findings suggest that antidepressant-treated groups had better compensatory responses that are associated with induction of hyperphagia, elevated body weight, and repletion of energy reserves leading to super abnormal linear growth23.Weight gain is a common outcome of antidepressant treatment in the clinical setting, but several studies have shown that chronic administration of various antidepressants results in failure to gain weight or “paradoxical” weight loss in rats, especially at high dosesTnf) and insulin sensitivity . Moreover, the expression of a gene involved lipolysis (Lipe) was significantly elevated in the FX and IM groups, indicating that there may have been enhanced lipolysis activity in these groups.We found that the obesity-prone FX animals had increased caloric intake, were lengthier and heavier, and had increased leptin levels, yet had significantly lower food intake ratio, suggesting that they had greater ability to store energy. The exacerbated weight gain in the FX group was associated with significant increase in lean mass, BMD, and BMC, and pronounced positive changes in bone cortical and trabecular morphology. Congruently, 9 months (endpoint) after stress and antidepressant use were ceased in the SADIO model, the obesity-prone FX subgroup had significantly greater body length in comparison to the obesity-prone NR and obesity-prone CS subgroups, implying that the propensity to gain weight can influence the rate of catch-up growth and bone allometry. On the other hand, although plasma leptin level was elevated in the obesity-prone FX subgroup in comparison to the obesity-prone CS subgroup, the obesity-prone FX subgroup did not have significant increase in fat mass; yet, the fat mass was significantly lower in the obesity-prone CS subgroup in comparison to the obesity-prone NR subgroup. Furthermore, the metabolic and lipid plasma profiles were not significantly elevated in the FX group, suggesting that there was no further metabolic/lipid dysregulation associated with FX-induced weight gain  resulted in increased body weight and length, accompanied by enhanced femoral bone features. Thus, the mechanism of SSRI action on bone after its discontinuation, in the presence of high-fat diet, is likely to be different from that of SSRI treatment alone, as our findings of elevated BV/TV and trabecular number are compatible with both reduced bone resorption, intensified bone formation (greater trabecular thickness) and increased cortical bone features and bone/body length suggesting enhanced bone growth. Regardless of treatment duration, antidepressant treatment most likely played a role during catch-up growth in the post-stress acclimation period. Furthermore, there was a strong correlation between plasma leptin level and BMC in the antidepressant-treated groups, which was absent in the NR or the CS control groups. Both, peripheral and central effects of leptin have been described to play a role in bone formation26. Peripheral leptin has been reported to have direct bone anabolic function and increase osteoblast number and activity27; however, via an indirect hypothalamic-mediated pathway, centrally leptin inhibits bone formation by regulating both phases of bone remodeling, resorption and, formation30, which suggest that short-term antidepressant treatment either lessened the central inhibitory and/or enhanced the peripheral role of leptin in bone formation in the SADIO model, resulting in enhanced bone features.The literature on the effect of long-term fluoxetine use on bone metabolism remains controversial. Ortuno et al.32. However, we found no significant differences in the endpoint IGF-1 plasma level between the groups; nevertheless, the secretion of GH declines with age and GH/IGF-1 levels may have been different in the earlier stage of weight gain34.Weight gain can be induced by mechanisms other than increased fat mass, and growth induced via the GH/IGF1; somatotropic axis is an important factor in weight gain. Wu et al. (2009) demonstrated that the over-expression of systemic IGF1 resulted in enhanced morphological bone features that resemble those of the SADIO model, such as increased cortical thickness and total bone mineral density, and enhanced trabecular bone volume35. Thus, weight gain of NR group could be due to diet-induced obesity rather than the growth associated with enhanced bone morphological features.In this study, we show that IGF1 mRNA levels in the liver are elevated in the CS group than in comparison to the NR group. Although there was no significant difference in systemic IGF-1 level between the groups, significantly lower IGF1 mRNA level in liver in NR group could provide explanation that NR group did not have enhanced bone morphological features compared to CS group. Sjögren et al. (2002) showed that inactivation of liver IGF1 induced a decrease in cortical cross-sectional area and periosteal circumference in the mid-diaphyseal region of the femur and was associated with weaker bone in the mechanical loading test36. While on the one hand, serotonin receptors 5-HT2A and 5-HT2c have been proposed to play a significant role in the inhibition of appetite and food intake37, on the other hand, long-term FX treatment has been shown to lead to weight gain38. Despite numerous studies, the effects of FX treatment in weight regulation remain elusive. Adherence to antidepressant treatment is low; in a large European study of 7525 patients, 56% abandoned treatment within 4 months, and weight gain was a major side-effect of antidepressant. Antidepressants including mirtazapine39, paroxetine40, and amitriptyline41 have been associated with long-term weight gain, and meta-analysis showed that amitriptyline and paroxetine induced the greatest weight gain in periods over 4 months;36 thus, future investigations could address the effects of these antidepressant drugs on weight regulation in the SADIO model.Previous findings have suggested that short-term FX treatment induces weight lossThere are several limitations and future directions for this study. Although our study indicated that stress and antidepressant (SAD)-induced weight gain is associated with growth, we could not clearly show whether the somatotropic axis is involved. We only measured GH/IGF-1 levels at the end of the experiment and as animals got older, the growth rate was sustained at much slower rate than in earlier periods. In the future, pulsatile GH levels in plasma should be characterised during an earlier period when a higher growth rate is observed, in order to obtain a better understanding of the mechanism behind SAD-related weight gain.In the SADIO model, 6 h of CRS unavoidably may have resulted in food deprivation. Although restraint stress was applied during the light cycle, the effects of food restriction experience on diet-induced weight gain and bone formation cannot be ruled out. For future studies, one additional group with 6 h of food deprivation should be included in the experimental design.45. Furthermore, Yirmiya R et al. (2006) have shown that chronic stress decreased osteoblast numbers and bone formation via activation of the sympathetic nervous system, and co-treatment with IM attenuated bone loss only in animals that improved from depressive-like behaviors (responders)45. However, to our knowledge this is the first report on the long-lasting effects of short-antidepressant treatment in body growth and bone features, which may manifest dissimilarly in subsets of the population with different propensity to gain weight while consuming high-fat diets. As osteoporosis is a condition with significant morbidity and increasing prevalence due to the aging population47, our findings demonstrating that environmental factors may modulate the role of leptin, may contribute to the understanding of aspects relevant to the heterogeneity and treatement approaches of this condition48.Our findings of decreased bone structural features after stress are compatible with those described for major depression and chronic stress-induced depressive-like behavior leading to bone lossThis study has several key findings: (1) Weight gain after recurrent stress and short-term antidepressant treatment followed by diet-induced obesity is different from that of the classic diet-induced obesity. (2) Short-term antidepressant treatment has long-term consequences, and ameliorated the effects of chronic stress on body growth (FX), had long lasting effects in anxiety-like behavior (FX) and adipose tissue gene expression (FX and IM), and affected bone allometric processes that lead to exacerbated bone growth  and enhanced bone structural features (FX). (3) Those effects on body growth and bone features are highly associated with leptin levels. (4) There is a significant interaction between short-term antidepressant treatment and leptin, that likely lessened the central role of leptin in inhibiting bone formation; thus, pharmacological agents may influence the role of leptin in long-term bone regulation. Clinical studies monitoring body weight, caloric intake, and body and bone features after antidepressant discontinuation are needed to confirm our observations in the clinical setting.Supplementary Information


Treatment success rates of rifampicin resistant (RR)/multi-drug resistant (MDR) tuberculosis (TB) in South Africa range from 43–48%, falling short of the World Health Organization’s target of ≥75%. We present rates and assess predictors of attrition by 12 months on treatment.Prospective observational cohort analysis of adults (≥18 years) initiating RR/MDR-TB treatment from 01 March 2013 to 30 September 2016. Attrition was defined as a combination of death and loss to follow-up  by 12 months on treatment. Predictors of attrition were identified using Cox Proportional Hazards models to estimate crude (HR) and adjusted hazard ratios (aHR) with corresponding 95% confidence intervals.By 12 months on treatment, 75/240 (31.3%) patients had either died  or been LTFU . Patients with moderate/severe anaemia , and those who were smear positive at baseline  were significantly more likely to die or be lost from care.At this outpatient DR-TB treatment site, there was a high rate of attrition halfway through the standard treatment course at 12 months of 31%. High rates of attrition by 12 months on treatment may continue during the second-half of therapy. South Africa, with the highest tuberculosis (TB) incidence in the world , in March 2012 South Africa implemented Xpert MTB/RIF , a rapid molecular assay used for the detection of mycobacteria tuberculosis (MTB) and RR as the first-line diagnostic test for TB, 4. FollPrior to 2012 all RR/MDR-TB cases were treated at specialist inpatient facilities for the duration of the intensive phase of treatment –9.In an attempt to reduce treatment initiation delays and treatment costs as well as to improve treatment access and outcomes, the South African National TB Programme (NTP), implemented a policy of “decentralized and deinstitutionalized treatment” of DR-TB in 2011. Under tThe parallel implementation of Xpert MTB/RIF and decentralization of treatment have resulted in a substantial increase in the numbers of patients accessing treatment and signWe have previously described the high rates of early mortality (9%) and LTFU (14%) by 6 months at a single decentralized DR-TB treatment site in Johannesburg, South Africa[We conducted a prospective observational cohort study of patients with RR-TB (regardless of resistance to other TB drugs) at an outpatient decentralized hospital-based TB clinic in Johannesburg, South Africa.We included adult patients (≥18 years) who initiated RR/MDR-TB treatment between 01 March 2013–30 September 2016. Complicated cases or patients with extensively drug-resistant TB (pre-XDR/XDR-TB)  were not eligible for the study and were referred to Sizwe Tropical Diseases Hospital for inpatient treatment. Patients who transferred to another facility during the first 12 months of treatment (no outcome available), were excluded from the outcomes analysis. Likewise, patients who had transferred-in to the study site after receiving ≥1 month of DR-TB treatment from another site as well as those with discordant laboratory results  were also excluded .The TB Focal Point (TBFP) is an outpatient TB clinic located within the tertiary-level, public academic Helen Joseph Hospital, in Johannesburg, South Africa. Under the South African NTP, the clinic became a decentralized DR-TB treatment facility providing comprehensive integrated HIV and TB care in 2012, 15.We used an in-house electronic patient medical record using data that is prospectively collected, regularly checked for accuracy and completeness, and managed on-site by clinic and study staff.Treatment outcomes were measured by 12 months post RR/MDR-TB treatment initiation. Due to the relatively small number of patients having experienced death or LTFU by this time, these outcomes were combined into our primary outcome, namely attrition by 12 months. Baseline demographic and clinical variables were evaluated as risk factors for attrition . We defia priori  or with a p-value <0.10 in crude analysis were included in the multivariate model. Schoenfeld residuals were used to test the assumption of proportional hazards. Adjusted models were assessed using complete case analysis. All analyses were carried out using SAS version 9.4 .The demographic and clinical characteristics of patients included in the analysis were presented using medians with corresponding interquartile ranges (IQR) for continuous variables while categorical variables were presented as simple proportions. Kaplan Meier survival analysis was used to determine the timing of death, LTFU and attrition in the first 12 months of treatment. Loss to follow-up was defined as treatment interruption ≥2 months from the last scheduled clinical visit, with no subsequent visit. Considering the potential time variance of the primary outcome observed factors associated with attrition were identified using Cox proportional hazards models to produce crude (HR) and adjusted hazard ratios (aHR) with corresponding 95% confidence intervals (CI). Person-time accrued from RR/MDR-TB treatment initiation until earliest of death, LTFU, or 12 months on treatment. Variables Ethical approval was granted by the Human Research Ethics Committee at the University of the Witwatersrand (protocol number: M130205). Patients provided written informed consent for study participation. The study is presented according to the STROBE checklist for observational cohorts.During the study period, 347 patients initiated RR/MDR-TB treatment and met study eligibility criteria. Of these, 14 (4.0%) did not provide informed consent, had incomplete consent documented or declined to participate, leaving 333 enrolled in the study. Twenty seven patients  with discordant RIF-resistance results who were subsequently treated as drug sensitive TB were excluded from the outcomes analysis as were 66 patients  who transferred to a different treatment facility and for whom 12-month outcomes could not be assessed. There remained 240 patients in the final analytic cohort .3 (IQR: 27.0–234.5) and more than half  having a CD4 cell count ≤100 cells/mm3 at RR/MDR-TB treatment initiation. Close to half of HIV positive patients were already on ART when starting RR/MDR-TB treatment , but 40% of the patients on ART for 6 months or more had a detectable viral load ). Median haemoglobin at start of treatment was 10.6 g/dL (IQR: 8.7–12.3), with close to 50% of patients having either moderate  or severe anaemia .The majority of patients were HIV co-infected , with a low median CD4 cell count of 88.5 cells/mmOver one third of patients in the cohort were diagnosed with RR/MDR-TB while hospitalized ; the rest were diagnosed at an outpatient clinic facility. Median time from diagnosis to treatment initiation was 9.0 days (IQR: 6.0–38.5) with less than half of patients initiating within 1 week . The proportion of positive smear microscopy results was slightly higher among those enrolled than in those included in the outcomes analysis .This study examined RR/MDR-TB treatment outcomes at a single decentralized facility in Johannesburg, South Africa 12 months after treatment initiation, approximately halfway through the prescribed standard 18–24 month long-course regimen in use at the time of the study. Twelve-month mortality in this cohort of patients was 15%. Loss to follow up was also substantial at 16%. Overall attrition thus reached 31% by 12 months post RR/MDR-TB treatment initiation. Our 12-month results are already similar to other studies from the region, where attrition by 24 months (combined outcome of death and LTFU) ranged from 20% to 46%, with variability between regions of the country and models of care delivery.We found that patients who had moderate or severe anaemia or who were smear-positive at treatment initiation to be more likely to experience death or LTFU. In adjusted analyses, moderate/severe anaemia was associated with a twofold increase in the risk of death or LTFU. This likely reflects the association between anaemia of chronic inflammation and advanced TB and HIV disease, 23. NumAlthough not reaching statistical significant in the crude or adjusted analysis, HIV infection in combination with ART, was also a risk factor for attrition. We did find that close to half of patients on ART for longer than six months at the time of RR/MDR TB treatment initiation had a viral load ≥1000 copies/ml consistent with virologic failure. The poorer immunological condition of these patients may then increase their risk of poor treatment outcomes.The highest risk of death in our cohort was during the first three months of RR/MDR-TB treatment, suggesting that there may be a role for closer monitoring of high-risk patients: those with advanced HIV, moderate/severe anaemia, or smear-positive disease, those whose diagnosis of RR/MDR-TB was made in a hospital setting or did not start RR/MDR-TB treatment within a week of diagnosis. Previous studies have demonstrated that decentralized and outpatient care for DR-TB has been crucial to improving access to treatment, reducing the time to treatment initiation, and reducing primary LTFU, 29. It While 12 months is an interim time point in the treatment course in use during this study, it is the endpoint of the 9–12 month short course recommendation made by the WHO in 2016, providiThis study was conducted at a single, urban treatment site in Johannesburg, South Africa with a high-rate of HIV co-infection. Findings reported here may not be representative of the entire RR/MDR-TB population in South Africa. We were not able to obtain outcomes for the substantial minority of patients who transferred to other facilities prior to completing 12 months’ follow-up, but outcomes for this group may differ from those in our analysis.A third of patients with RR/MDR-TB (31%) in Johannesburg, South Africa died or were LTFU by 12 months after RR/MDR-TB treatment initiation. Early attrition was related to baseline disease severity, namely moderate and severe anaemia and smear positive pulmonary disease. High rates of attrition by 12 months, the halfway point of RR/MDR-TB treatment, suggest that earlier monitoring, before the 24-month regimen endpoint, can alert clinicians to attrition risks while patients remain in care. High rates of death and LTFU at 12 months also suggest that attrition will remain unacceptably high during the newer short-course DR-TB treatment regimen endorsed by the WHO in 2016 unless it is accompanied by interventions targeting early death and LTFU.


HIV Self-Testing (HIVST) aims to increase HIV testing coverage and can facilitate reaching the UNAIDS 90-90-90 targets. In Senegal, key populations bear a disproportionate burden of HIV and report limited uptake of HIV testing given pervasive stigma and criminalization. In these contexts, HIVST may represent a complementary approach to reach populations reporting barriers to engagement with existing and routine HIV testing services. In this study, 1839 HIVST kits were distributed in Senegal, with 1149 individuals participating in a pre-test questionnaire and 817 participating in a post-test questionnaire. Overall, 46.9% (536/1144) were first-time testers and 26.2% (300/1144) had tested within the last year; 94.3% (768/814) reported using the HIVST, and 2.9% (19/651) reported a reactive result which was associated with first-time testers (p = 0.024). HIVST represents an approach that reached first-time testers and those who had not tested recently. Implementation indicators suggest the importance of leveraging existing community structures and programs for distribution. Increasing coverage of HIV testing and early detection of seroconversion among people living with HIV is essential for effectively responding to the HIV pandemic. Early detection of HIV and initiation of antiretroviral therapy (ART) significantly reduces HIV-related morbidity and mortality, and can improve the quality of life for people living with HIV while also eliminating the risk of onward HIV transmission –3. SimilHIVST can potentially overcome barriers to HIV testing uptake and accessibility by placing the locus of control of testing on the individual, increasing confidentiality, and allowing members of marginalized and stigmatized groups to test in settings of privacy, safety, and with dignity . Oral HIThe West African country of Senegal is one of the countries in sub-Saharan Africa where an HIVST policy is currently under development . SenegalGiven the HIV epidemic profile in Senegal and the limited uptake of HIV prevention and treatment services among key populations in the country, HIVST may represent an impactful strategy for increasing the uptake and coverage of HIV testing and accelerating progress towards achieving 90-90-90 goals. This study aimed to assess the acceptability of HIVST for key populations and people in their social and sexual networks and secondly, to assess the effectiveness of HIVST in reaching first-time testers. These results will inform appropriately scaled implementation of HIVST in Senegal and across West Africa.This is a pilot study which distributed HIVST kits through targeted venues and recruited individuals through convenience sampling to participate in pre and post HIVST socio-behavioral questionnaires.OraQuick HIV Self-Test Kits  were distributed to individuals in Dakar and Ziguinchor through venue and social network-based distribution. The HIVST kits included an OraQuick test device, written and pictorial step-by-step instructions, supplementary information on the test and HIV, and a referral card with information for confirmatory testing sites and study contacts. Instructions and supplementary information were provided in French and Wolof and adapted to the Senegalese context.HIVST kit distribution and participant recruitment was led by study partner, Enda Santé, and aimed to reach populations with increased vulnerability of HIV acquisition and high levels of health care related stigma, including MSM, FSW, PWID, and clients of FSW. .The venue-based approach for distribution and recruitment utilized directly assisted distribution of HIVST and was conducted through outreach to sex work venues, bars, nightclubs, hot spots, and mobile clinics, as well as health facilities that provide services to key populations. Venues were selected based on recommendations of community partners with previous experience in the communities, and leveraged existing programmatic activities. Directly assisted distribution of HIVST followed the WHO definition  and was A small sample of additional HIVST kits were distributed through social network-based unassisted distribution. The social network-based approach was focused on providing a primary recipient with one HIVST kit for themselves and two additional kits to distribute to individuals within their network. Social network-based distribution leveraged venue-based distribution to engage the primary HIVST recipient, who received the HIVST kits directly from the trained distributor. The primary recipient then distributed to secondary recipients through indirect, unassisted distribution as defined by WHO . SecondaConvenience sampling was used to recruit individuals into the study at the time of HIVST kit distribution. Individuals receiving the HIVST kits through directly assisted venue-based distribution were asked if they wished to participate in a pre- and post-test survey. Data from social network-based distribution were only obtained from the primary recipient as follow up was not possible for the network-based HIVST kit recipients. Participants were eligible if they reported being 18 years of age or older; capable of and willing to provide informed consent; agreed to use the HIVST; and spoke Wolof and/or French. Participation was voluntary, and individuals could receive an HIVST kit regardless of survey participation. All pre- and post-test surveys were administered to eligible participants by trained interviewers. Among consenting participants, an interviewer administered pre-test surveys at the distribution site before HIVST utilization. Pre-test surveys captured information on demographic characteristics, HIV risk behaviors, HIV testing history, and motivation for testing.Among individuals who opted to test at the HIVST distribution sites, the HIVST was collected through a test disposal box after self-administration and was read immediately. The result was logged to track the overall results observed, but not connected to the individual participant. This approach was used to compare aggregate level results to those self-reported in the post-tests. Post-test surveys assessing self-reported HIVST use and acceptability were conducted by phone two weeks after the HIVST kit distribution. Data were not obtained from secondary recipients.Ethical review and approval were provided by the National Research Ethics Committee in Senegal and the Johns Hopkins School of Public Health Institutional Review Board.Key population characteristics were self-reported. Sex worker was defined as reporting exchanging sex for money or goods, and with more than half of income being from selling sex in the past 6 months. Male sex workers (MSW) were defined as sex workers above, as well as being assigned male sex at birth; and FSW were defined as sex workers as above and assigned female sex at birth. MSM was defined as being assigned the male sex at birth and ever having oral or anal sex with another man. Transgender women were defined using a two-step gender assessment of reporting male sex assigned at birth and gender identification as a woman. PWID were defined as ever having injected illicit drugs. Key population categories were not mutually exclusive. Key population was defined as meeting the criteria of at least one of the six key population categories.First-time testers were defined as individuals who self-reported never having received an HIV test prior to the pre-test questionnaire. HIVST reactivity results were collected in two ways: 1. Results collected from used HIVST at the distribution sites; and 2. Self-reported HIVST results from those who participated in the post-test phone survey. Acceptability measures were informed by The Society for Implementation Research and Collaboration Indictor Review, however, have not yet been validated .Demographic characteristics and HIV testing history were determined from pre-test questionnaires. Logistic regression was used to assess the crude relationship between HIV testing history (first-time vs. previous testers), demographic characteristics, and HIV risk behaviors. Multiple multivariable logistic regression models were developed to separately assess each demographic characteristic, HIV testing history, HIV risk behaviors as primary predictors of first-time testers and adjusted for a priori demographic characteristics. Pearson’s Chi squared tests were used to assess the crude relationships between first-time testers and HIVST use and acceptability, as well as the relationships between self-reported HIVST result and use and demographic characteristics. A significance value of p < 0.05 was used for all analyses.A total of 1839 HIVST kits were distributed between April 2017 to June 2018, and 62.5% (1149/1839) of recipients participated in the pre-test questionnaire before receiving the HIVST  had received additional HIVST kits for secondary, unassisted distribution, of which 80.0% (36/45) distributed the additional HIVST kits. Among individuals not provided additional HIVST kits for unassisted distribution, 1.2% (9/730) gave their HIVST kit to someone else.Among participants who completed the pre-test, 47.9% 539/1125) were in Dakar and 52.1% (586/1125) were in Ziguinchor  self-reported membership of a key population group with 18.8% (204/1085) sex workers specifically, 14.3% (155/1085) FSW and 4.4% (48/1085) MSW; 15.1% (174/1149) MSM; 3.7% (42/1131) PWID; and 1.7% (20/1148) transgender women.Among pre-test respondents, 46.9% (536/1144) of participants were first-time testers, 26.9% (308/1144) had ever tested for HIV but not within the last 12 months, and 26.2% (300/1144) had tested within the last 12 months.Among key populations, 36.8% (136/370) were first-time testers  were first-time testers, and in Ziguinchor 52.6% (306/582) were first-time testers  compared to Dakar  had been worried about their HIV status, which was negatively associated with being a first-time tester   reported using the HIVST of which 43.5% (333/765) were first-time testers  participants reported being comfortable using the HIVST. In total, 86.1% (576/669) found the instructions easy to follow, and 94.4% (638/676) thought their family of friends would use the HIVST. After receiving the HIVST, 30.6% (244/797) discussed HIV testing with a sexual partner or friend. Among participants 78.5% (307/391) would be comfortable asking a primary sexual partner to use an HIVST, and 65.8% (150/228) would be comfortable asking a casual sexual partner to use an HIVST.Among post-test respondents reporting a reactive result, 42.1% (8/19) used the test on site, and 57.9% (11/19) used the HIVST at home Table . Among tThis study demonstrates that HIVST can effectively engage first-time testers at risk for HIV in Senegal, including key populations, cisgender men, and young adults. Expanding access to HIVST may increase the coverage and frequency of HIV testing and thus have an important role in linking people living with HIV to diagnosis and treatment services and potentially mitigating the HIV epidemic in Senegal. Overall history of HIV testing as well as frequency of testing remains low among key populations, as well as among young adults in their social and sexual networks in Senegal. HIVST result reactivity was associated with first-time testing, and among those who tested with an HIVST, acceptability was high for both first-time testers and those reporting previous HIV testing. However, consistent with some earlier studies, confirmatory testing and linkage to care was a challenge during the implementation of HIVST in Senegal , 27.This study highlights that HIVST was able to reach a large proportion of individuals, and in particular key populations, who had never received an HIV test as well as those who had not tested recently. Notably, approximately half of MSW, MSM, PWID, and transgender women reached through HIVST reported not having tested for HIV. Few programs currently exist to provide tailored health services to PWID and transgender women in Senegal, and this study suggests that HIVST may provide an opportunity for PWID and transgender women to increase uptake of testing in this context . The proThis small scale implementation of HIVST leveraged existing programs and networks working with key populations to distribute HIVST. Despite available services and programs in Senegal, HIVST was able to reach a large proportion of first-time testers in this study. Therefore, HIVST represents a promising new approach to increase coverage and uptake of HIV testing through leveraging current programs. However, adoption and integration of HIVST into existing programs will require a revision of the current HIV testing targets for programs in Senegal. HIVST indicators have been incorporated into the PEPFAR Monitoring, Evaluation, and Reporting (MER 2.0) Indicator Reference Guide representing appropriate indicators for collection in HIV testing programs . NotablyFirst-time testers were associated with HIVST result reactivity in this study, with the majority of self-reported reactive results being among first-time testers. These findings suggest the potential effectiveness of HIVST in increasing HIV diagnosis among those living with HIV in Senegal and not accessing traditional testing services. Additionally, acceptability was overall high among individuals who participated in the post-test survey, as shown in other settings , 31, 32.Although acceptability of HIVST has been high in other studies, consistent evidence on confirmatory testing and linkage to care similarly remain sub-optimal , 27. In Several limitations should be considered in this study. Participation in the pre- and post-test questionnaires was voluntary and may not represent the full sample of individuals who participated in HIVST distribution. The results may therefore be subject to bias. Participants who received HIVST through network distribution were not captured in data collection and are not represented in this analysis. Disclosure of key population status as well as positive reactivity from the HIVST were low in self-reported measures of this study. The distribution strategy prioritized members of key populations and worked closely with existing programs providing services to these populations. However, only one-third of the study sample self-reported key population status. Therefore, it may be that HIVST reached individuals who may not currently be at high risk of HIV, in which case there is a need to consider strategies to more effectively target key populations. Alternatively, key population status may have been underreported, in which case HIVST was able to reach individuals unwilling to disclose their key population-related behavior and less integrated into the key population networks . AdditioIn Senegal, key populations bear a disproportionate burden of HIV, and report limited uptake of existing HIV testing services given pervasive stigma and criminalization. In these contexts, HIVST may represent a complementary approach to reach populations reporting barriers to engagement with existing and routine HIV testing services. These data suggest the potential impact that HIVST could have in complementing existing HIV testing services by reaching a diverse group of first-time HIV-testers as well as those who have not tested recently in Senegal. This small-scale implementation further suggested the importance of leveraging existing structures and programs for distribution. Moreover, since HIVST has the potential to disrupt traditional testing approaches, sustained engagement with government and community stakeholders is needed to inform optimal implementation strategies of HIVST.


It is well established that physical activity (PA) protects against mortality and morbidity, but how long-term patterns of PA are associated with mortality and cardiovascular disease (CVD) remains unclear.3231 men recruited to the British Regional Heart Study, a prospective cohort study, reported usual PA levels at baseline in 1978–1980 (aged 40–59 years) and at 12-year, 16-year and 20-year follow ups. Twenty-year trajectories of PA, spanning from 1978/1980 to 2000, were identified using group-based trajectory modelling. Men were subsequently followed up until 30 June 2016 for mortality through National Health Service central registers and for non-fatal CVD events through primary and secondary care records. Data analyses were conducted in 2019.Three PA trajectories were identified: low/decreasing (22.7%), light/stable (51.0%) and moderate/increasing (26.3%). Over a median follow-up of 16.4 years, there were 1735 deaths. Compared with the low/decreasing group, membership of the light/stable  and moderate/increasing  groups was associated with a lower risk of all-cause mortality. Similar associations were observed for CVD mortality, major coronary heart disease and all CVD events. Associations were only partially explained by a range of confounders. Sensitivity analyses suggested that survival benefits were largely driven by most recent/current PA.A dose-response relationship was observed, with higher levels of PA from midlife to old age associated with additional benefits. However, even fairly modest and sustained PA was protective and may be more achievable for the most inactive. Physical activity (PA) is inversely associated with risk of all-cause mortality and cardiovascular disease (CVD) events in old age.Furthermore, conventional methods for defining PA changes are theory driven, which typically involves dividing subjects into clinically meaningful groups based on a relevant cut point .Two previous studies using GBTM suggested that more active trajectories are associated with a reduced risk of all-cause, cancer-related and CVD-related mortality.The British Regional Heart Study (BRHS) is a prospective cohort study involving 7735 men (aged 40–59 years at baseline) recruited from primary care practices in 24 towns in Great Britain between 1978 and 1980.1)) were taken by nurses. Information on measurement techniques are described in the supplementary files (online 2 or overweight/obese: BMI ≥25.0 kg/m2).At the 20-year follow-up, participants attending the physical examination provided a fasting blood sample, which was analysed for total, high-density lipoprotein (HDL) and low-density lipoprotein cholesterol (LDL), insulin, von Willebrand factor (vWf), interleukin 6 (IL-6), n-terminal pro-brain natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin T (Hs-TnT). Other physical measurements including waist circumference, blood pressure and lung function  and sport/exercise. Questions and response options were virtually identical at each wave. Responses were scored based on the intensity and frequency of the activity.Men were followed up for major CVD events and mortality from any cause from the 20-year follow-up in 2000 until 30 June 2016. Information on cause of death was collected through the tagging procedures of the National Health Service Central Registers in Southport for England and Wales, and in Edinburgh for Scotland ). Information on non-fatal events was obtained from general practitioners and biennial reviews of the patients’ medical records. For major CHD events , fatal MI was defined as ICD-9 codes 410–414 and non-fatal MI was defined as heart attack or coronary thrombosis in accordance with the WHO diagnostic criteria. For major stoke events , fatal stroke was defined as ICD-9 codes 430–438 and non-fatal stroke events included those that caused a neurological deficit for >24 hours. Fatal CVD was defined as ICD-9 codes 390–459. Outcome measures included all-cause and CVD mortality, major CHD events , major stroke events  and all CVD events .GBTM was used to identify 20-year PA trajectories over the four waves. Models were conducted using the Stata TRAJ plugin,1. Model 3 additionally adjusted for the inflammatory and haemostatic markers IL-6 and vWf. Model 4 additionally adjusted for the cardiac markers Hs-TnT and NT-proBNP.Cox proportional hazards models were used to estimate associations between trajectory groups and all-cause and CVD mortality, major CHD, stroke and all CVD events. For participants with no events, data were censored on the 30 June 2016. The proportional hazards assumption was examined using graphical methods and Schoenfeld residuals. Model 1 adjusted for age, marital status, alcohol consumption, smoking status and previous diagnosis of MI, stroke and diabetes (reported at the 20-year follow-up), and occupational class and region (reported at baseline). To examine the role of potential mediators, model 2 additionally adjusted for metabolic CVD risk factors measured at the 20-year follow-up, including LDL, HDL, systolic blood pressure, insulin, waist circumference and FEVAs CVD events can influence PA and increase the risk of mortality, trajectories were also identified after excluding participants with CVD at the 20-year follow-up and associations with subsequent outcomes were estimated. Furthermore, as undiagnosed chronic conditions, such as CVD , may initiate changes in PA and increase the risk of mortality, analyses were performed after excluding the first 2 years of follow-up, thus minimising the possibility of reverse causation. As well as using GBTM to identify trajectories, we also grouped subjects based on observed trajectories using binary exposure measurements from baseline through 12-year and 20-year follow ups. The PA score was collapsed into a binary variable at each of the three time points, with 1 indicating at least ‘light’ PA levels and 0 denoting ‘inactive’ or ‘occasional’ activity levels. This cut point has previously been used in studies using data from this cohortFrom the 7735 men recruited at baseline, 5516 survived the next 20 years of follow-up, of which 4252 attended the 20-year physical examination (77% of survivors). Of these, 197 men with <3 PA measures were excluded and a further 824 men were excluded due to missing covariate data, leaving 3231 available for analyses. Compared with the final sample, excluded men (n=4504) were significantly older at baseline , were more likely to be inactive , come from manual occupations  and suffer from a range of health conditions, including breathlessness , overweight/obesity , arthritis  and bronchitis .Three trajectory groups emerged as the most parsimonious description of the longitudinal data: low-decreasing (22.7%), light-stable (51.0%) and moderate-increasing (26.3%) see . ResultsOver a median follow-up of 16.4 years, there were 1735 deaths. As shown in In several models, the proportional hazards assumption was not met for age. Where violated, age was collapsed into 10 categories and centred. Models were run again with the categorised age variable, but no meaningful differences were observed (data not shown). All associations were similar after removing men with pre-existing CVD see  and the 44% (n=1312) of men were classified as persistently active (1-1-1), while membership of the other groups ranged from 5% to 13%. Lower unadjusted mortality rates were observed for the more active trajectories see . The higPatterns of PA from midlife to old age were associated with all-cause and CVD mortality and major CHD and CVD events but not major stroke events. A moderate-increasing pattern was associated with the lowest risk of the aforementioned outcomes, but a light-stable pattern was also associated with significant benefit, which may be more achievable in the most inactive. Furthermore, adjustment for a range of confounders and mediating risk factors only partially explained these associations, suggesting that there may be other underlying biological pathways involved. For instance, physical activity may improve endothelial function and fibrinolytic activity; it may also have a direct effect on the cardiovascular system.It is well established that higher levels of PA are associated with a lower risk of mortalityUsing GBTM, we did not identify groups of older adults that made meaningful changes in PA, highlighting that adults shifting from low to even modest levels of PA are rare. Instead, the GBTM trajectories suggest that prior PA dictates PA in old age. This is consistent with some other GBTM studies using continuous measures of PA that have also identified trajectories that do not typically overlap during this life period.1 and device-measured PA, self-reports may still be subject to recall and social desirability bias.Studies using data-driven methods to identify PA trajectories and examine associations with subsequent mortality are scarce. One such study also identified three PA trajectories over a 7-year follow-up in an older sample.During the transition to old age, PA trajectories were associated with all-cause and CVD mortality and major CHD and CVD events. A dose–response relationship was observed, with a moderate-increasing pattern of PA providing the most protection against mortality and CVD, but even a sustained light level of PA was beneficial and may be more feasible for inactive adults.Higher levels of physical activity (PA) are associated with a reduced risk of all-cause mortality and major cardiovascular disease events in old age.There is some evidence that long-term changes in PA during adulthood can impact the risks of mortality and cardiovascular disease (CVD), but the conventional approaches used to define how PA changes may be inadequate for describing the true patterns of PA over time.Moderate increasing levels of PA from midlife to old age were associated with the lowest risk of major coronary heart disease events, CVD mortality and all-cause mortality in later life.However, a more modest level of PA that is sustained across adulthood may be sufficient to reduce the risk of these outcomes in later life.Promoting a light sustained level of PA across adulthood may be a feasible intervention target for the most inactive and provides similar benefits as greater volumes of PA across the adult life course.


Women living with HIV (WLHIV) have an increased risk of malaria in pregnancy (MiP). It is unclear if MiP in WLHIV causes a systemic inflammatory response and increases the risk of adverse birth outcomes, especially for women receiving antiretroviral therapy (ART) and daily trimethoprim-sulfamethoxazole (TMP/SXT). We analyzed repeated plasma samples in a cohort of malaria-exposed Ugandan WLHIV receiving ART and daily TMP/SXT to examine changes in inflammatory markers across pregnancy and their association with birth outcomes. Concentrations of CHI3L1, CRP, IL-18BP, IL-6, sICAM-1, and sTNFR2 were quantified by ELISA in 1115 plasma samples collected over pregnancy from 326 women. MiP was associated with increased sTNFR2, sICAM-1 and IL-18BP concentrations across pregnancy. Women who delivered preterm had elevated concentrations of sTNFR2 and altered levels of IL-6 during pregnancy. Women with sTNFR2 concentrations in the highest quartile within 6 weeks of delivery had an increased relative risk of preterm birth. Our results indicate that despite daily TMP/SXT, MiP in WLHIV induced a systemic inflammatory response that was associated with an increased risk of preterm birth. These findings highlight the need for additional strategies to protect WLHIV from malaria infection in pregnancy to promote healthy outcomes for mother and child. Cultural, social, and economic inequality has resulted in women of reproductive age representing almost half of all adults living with HIV worldwide2. Increased use of WHO-recommended multi-drug antiretroviral therapy (ART) during pregnancy has improved health outcomes in women living with HIV (WLHIV). Rates of pregnancy in WLHIV are comparable to the general population5; however, risk of adverse birth outcomes, including preterm birth (PTB), small-for-gestational age, low birth weight, and stillbirth, remain higher in WLHIV6, irrespective of ART2.Two thirds of all people living with HIV reside in sub-Saharan Africa. In sub-Saharan Africa, HIV and malaria co-infection poses an important health risk for pregnant women. Young women (age 15–24 years) make up 11% of the adult population and 20% of new HIV infectionsPlasmodium falciparum malaria in pregnancy (MiP)7. Pregnant women are more likely to be infected with malaria and to experience more severe disease9. Women with MiP are at higher risk for adverse birth outcomes10. Recommended care for pregnant women living in malaria-endemic regions includes the use of insecticide-treated bed nets (ITNs), intermittent preventive treatment of malaria in pregnancy , and clinical management of MiP and anemia. However, low rates of ITN coverage, increasing resistance to IPTp drugs, and limited access to antenatal care are important barriers to impact, and MiP remains a global health priority.The risk of a poor birth outcome is increased for pregnant women living in malaria-endemic regions. An estimated 85 million women a year are at risk of 11. Co-infection with malaria and HIV is associated with an increased risk of PTB and low birth weight compared to the risk from either infection alone11. An estimated 15 million children are born preterm every year, resulting in over one million deaths in children under the age of five12. The majority of all PTBs - over 60% - occur in sub-Saharan Africa and South Asia. This high prevalence of PTB coincides with high disease burden in pregnancy, including the risk of HIV and malaria infection. An enhanced understanding of the systemic response to co-infection during pregnancy may inform new interventions to reduce adverse birth outcomes, including PTB.For many sub-Saharan African women of reproductive age, co-infection with HIV and MiP further increases the risk of poor birth outcomes. WLHIV have an increased incidence and density of peripheral and placental malaria infection, and experience more complications including maternal anemia and maternal death13. Here we examined changes in inflammatory markers over the course of pregnancy in a cohort of WLHIV in Uganda who were receiving ART as well as daily prophylactic treatment with trimethoprim-sulfamethoxazole (TMP/SXT). Due to its burden of disease in sub-Saharan Africa and the limited data on PTB in women at risk for MiP, PTB was our primary outcome of interest. We examined systemic inflammation across pregnancy in association with MiP and the risk of PTB.Systemic inflammation is a leading risk factor for spontaneous PTB3, and median HIV RNA viral load was 15.5 (IQR 2.4–59.4) log10 copies/mL (Table All women were ART naïve at enrollment before randomization to receive Lopinavir/ritonavir (LPV/r)- or Efavirenz (EFV)-based ART. No differences in demographics were observed between treatment arms  samples were tested per participant  and soluble TNFR II (sTNFR2). Concentrations of the soluble receptors are considered markers of TNF-mediated pro-inflammatory activity21. IL-18BP is an antagonist of pro-inflammatory IL-18. Levels of IL-18BP increase in response to elevated levels of circulating IL-18. The soluble adhesion molecule sICAM-1 is cleaved in response to inflammation and endothelial activation. Elevated circulating concentrations of sTNFR2, sICAM-1, and IL-18BP have been associated with various infections, as well as disease severity23. The elevated concentrations of sTNFR2, sICAM-1, and IL-18BP we observed in association with MiP in WLHIV, indicate a malaria-induced systemic inflammatory response. These findings are consistent with studies of MiP in HIV-uninfected women24.Here we report increased circulating levels of sTNFR2, sICAM-1, and IL-18BP in WLHIV with MiP. We focused on sTNFR2 and IL-18BP concentrations, as these proteins have been shown to be more informative indicators of systemic inflammation than TNF or IL-1825. Several lines of evidence link altered pro-inflammatory cytokine levels to the initiation of PTB, including the induction of myometrial contractions, preterm premature rupture of membranes (PPROM), and cervical maturation25. Here we observed altered kinetics of circulating IL-6 and sTNFR2 across pregnancy in women who subsequently delivered preterm. Pro-inflammatory factors including IL-6 are involved in regulating the intrauterine inflammatory response that contributes to fetal inflammatory response syndrome, membrane rupture, and PTB26. However, many studies examining the role of IL-6 in PTB have measured IL-6 only later in pregnancy 25. Our data provide some of the first evidence for a maternal systemic IL-6 response in association with PTB in WLHIV. In our cohort, the median concentration of IL-6 early in pregnancy was initially lower in women who went on to deliver preterm, but then exceeded term pregnancy concentrations after approximately 26 weeks of gestation. Since women in spontaneous term labour have elevated circulating IL-629, our data are consistent with a hypothesis that systemic inflammation can trigger premature initiation of physiological mechanisms leading to labour and delivery. Considerable evidence also links TNF to the pathology of PTB, including increased TNF concentrations in women in preterm labour and women with PPROM, as well as mechanistic roles for TNF in mediating membrane rupture, cervical ripening and infection-induced PTB25. In this cohort, MiP in WLHIV was associated with increased circulating levels of sTNFR2, IL-18BP, and sICAM-1 longitudinally. These findings are supported by similar observations in HIV-negative women, including an association between spontaneous PTB and elevated plasma sTNFR2 and sICAM-1 concentrations20. Although elevated IL-18BP has previously been associated with PTB20, it was not observed in this population. These data suggest that MiP-induced elevations in pro-inflammatory markers such as sTNFR2, IL-18BP, and sICAM-1, could disrupt the closely regulated balance of pro-and anti-inflammatory proteins required to maintain ongoing pregnancy, and thereby initiate pathways leading to PTB.Infections and the resulting inflammation are well-established risk factors for spontaneous PTB, although the precise pathobiology leading from inflammation to PTB is not fully understood30. The addition of an effective antimalarial to daily TMP/SXT may improve protection against MiP33; however, the appropriate antimalarial and dosing regimen for pregnant WLHIV remains controversial. It is possible that women in this cohort had malaria infection prior to enrollment. However 64% reported using TMP/SXT prophylaxis prior to enrollment and the majority of participants were receiving prophylactic treatment throughout pregnancy14. Collectively the available evidence is insufficient and additional studies are required to determine a drug-based strategy to adequately prevent MiP in WLHIV and promote healthy birth outcomes for mother and child.Notably, our results indicate that despite receiving ART and daily chemoprophylaxis with TMP/SXT, WLHIV had evidence of MiP together with systemic inflammation  across pregnancy and an increased risk of PTB. These data suggest that current WHO-recommended policies to prevent MiP may not provide optimal protection for WLHIV. While Tororo, Uganda is considered a high-transmission region for malaria, the prevalence of malaria in this study cohort was relatively low; however, women are still showing MIP-induced changes in inflammatory profiles. WHO recommends daily co-trimoxazole chemoprophylaxis during pregnancy for WLHIV to prevent malaria and opportunistic infections34.To our knowledge no previous study has reported longitudinal inflammatory profiles across pregnancy in a cohort of WLHIV at risk of MiP. Longitudinal sample collection allowed for the examination of changes in systemic inflammation in association with gestational age and malaria-infection status across pregnancy. This study had several strengths including the prospective design of the parent study, longitudinal collection of plasma samples, and the ability to control for multiple covariates, however there are also limitations. This is a retrospective analysis of a cohort of women enrolled in a randomized controlled trial. In the absence of an HIV-negative control group, it is not possible to examine the influence of HIV status on our observed systemic inflammatory response. We did not observe a direct association between MiP and PTB which may reflect the timing of malaria infection and diagnosis in pregnancy. In this study the majority of malaria diagnoses were made at delivery through histopathological examination of the placenta. In this study antenatal diagnosis was confirmed by blood smear and not PCR. Since blood smears are less sensitive than PCR, it is possible that they did not detect early malaria infections that could have triggered the persistent systemic inflammatory response associated with PTB that we observed. Given the small number of women with antenatal smear-positive malaria (n = 25) it was not possible to link birth outcome to the timing of malaria infection. Grouping all malaria infections may mask the effect that timing of infection has on the association between MiP and PTB. Further, as evidenced by the observation that changes in IL-6 were associated with PTB but not MiP, other factors for which we did not have data (i.e. intrauterine infection) could also have contributed to the association between systemic inflammation and PTB. Additional studies are required to examine longitudinal changes in inflammatory response in both HIV seropositive and seronegative women at risk of MiP. Further research is also required to establish how the timing of malaria infection during pregnancy impacts persistent systemic inflammatory responses and the risk of PTBCollectively our results suggest that MiP in WLHIV initiates inflammatory pathways that can increase the risk of PTB. These findings are consistent with mechanistic studies linking inflammation to PTB and add to a growing body of evidence that malaria-induced systemic inflammation is associated with poor birth outcomes, including PTB. These findings indicate that current treatment strategies for WLHIV may be inadequate to prevent MiP and the resulting systemic inflammatory responses and associated adverse birth outcomes.14. HIV positive, ART-naïve women who were ≥16 years of age and pregnant  were invited to participate. Women were eligible for enrollment at any CD4 cell count. Women who had received ART within the last 24 months were ineligible. Participants were randomized (1:1), following stratification by gravidity and gestational age at enrollment to receive LPV/r- or EFV-based ART. All participants received daily TMP/SXT (160 mg/800 mg). Blood collection took place at enrollment and all antenatal visits. MiP was diagnosed by blood smear at antenatal care visits, and at delivery by thick blood smear, PCR, malaria rapid diagnostic test, and by placental histology. The procedures for preparation and examination of placental specimens have been reported in detail14. All placental specimens were collected within 30 minutes of delivery. At this time a thick blood smear was collected, a rapid diagnostic test  was performed, and a dried blood spot was collected for PCR. Placental histology was performed on a 2 × 2 full-thickness biopsy embedded in paraffin wax and sectioned into slides stained with hematoxylin-eosin (H&E) and Giemsa. A trained investigator performed all histopathological analysis, which included an examination of malaria parasites, hemozoin pigment in intervillous fibrin, and macrophages in accordance with standardized criteria14. A composite measure of MiP included any evidence of malaria infection in pregnancy by antenatal peripheral blood smear and/or at delivery identified by placental histology, PCR, blood smear and/or rapid diagnostic test. Standardized assessments were completed at delivery including gestational age and birth weight . PTB is defined as delivery prior to 37 completed weeks of gestation, as confirmed by ultrasound dating. Women were eligible for inclusion in the present study if they had a singleton pregnancy with known birth outcome, and samples collected within 6 pre-specified gestational age bins: 16– < 20, 20– < 24, 24– < 28, 28– < 32, 32– < 36, 36– < 37 weeks of gestation.This was a secondary analysis of women enrolled in an open-label, single-site, randomized control trial of LPV/r-based versus EFV-based ART conducted in Tororo, Uganda from December 2009 to March 2013http://www.health.go.ug/docs/ucg_2010.pf). The study received ethical approval from Makerere University School of Medicine , the Uganda National Council for Science and Technology , the National Drug Authority , the University of California-San Francisco , and the University Health Network . The trial was registered at ClinicalTrials.gov (identifier:NCT00993031). Signed informed consent was obtained from each participant enrolled in the clinical trial to allow for blood collection and subsequent analysis of plasma samples for inflammatory proteins.All women received standard antenatal care according to the Ugandan Ministry of Health Guidelines . Maternal peripheral plasma samples were collected in EDTA and stored at −80 °C unthawed until testing. Plasma samples were processed using enzyme-linked immunosorbent assays  listed with dilution factors: CHI3L1 (1:1000), CRP (1:8000), IL-18BP (1:20), IL-6 (1:20), sICAM-1 (1:500), and sTNFR2 (1:200). The markers were selected based on previous studies reporting an association with MiP and PTB36. Descriptive data were summarized using median [IQR] or n (%). Baseline characteristics were compared between trial arms using the Mann-Whitney U test, Pearson Chi-square test, or Fisher’s exact test where appropriate. LME modeling 37 was used to examine longitudinal changes in loge-transformed protein concentrations across gestation by trial arm, malaria status, and PTB outcome. Loge–transformation was used as the concentrations of biomarkers showed deviations from normality (Shapiro-Wilks p < 0.05). The model included a random-intercept for each participant and a by-participant random slope for the effect of gestational age. We shifted the gestational age covariate to provide a meaningful intercept by subtracting the lowest gestational age from all gestational age values. All models included an interaction term between gestational age and treatment arm. Models adjusted for gestational age at blood sample collection, maternal age, body mass index (BMI: kg/m2), gravidity, and the interaction between treatment arm and gestational age. Visual inspection of residual plots did not reveal deviations from normality. Log-linked binomial regression models were used to estimate RR (with 95% CI) of malaria by immunological status , and PTB across quartiles of protein concentrations, with the lowest quartile as the reference category. In cases where the model did not converge, log-linked poisson regression models were used. RR of PTB by protein concentration in quartiles were estimated using the blood sample collected closest to the delivery date (up to a maximum of six weeks prior to delivery). All p-values were two-sided and values of <0.05 were considered statistically significant.Statistical analysis was performed using STATA v12  and R version 3.5.1  softwareSupplementary Information