Previously, transplantation of ovaries from young cycling mice into old postreproductive-age mice increased life span. We anticipated that the same factors that increased life span could also influence health span. Female CBA/J mice received new (60 d) ovaries at 12 and 17 months of age and were evaluated at 16 and 25 months of age, respectively. There were no significant differences in body weight among any age or treatment group. The percentage of fat mass was significantly increased at 13 and 16 months of age but was reduced by ovarian transplantation in 16-month-old mice. The percentages of lean body mass and total body water were significantly reduced in 13-month-old control mice but were restored in 16- and 25-month-old recipient mice by ovarian transplantation to the levels found in six-month-old control mice. In summary, we have shown that skeletal muscle mass, which is negatively influenced by aging, can be positively influenced or restored by reestablishment of active ovarian function in aged female mice. These findings provide strong incentive for further investigation of the positive influence of young ovaries on restoration of health in postreproductive females. In many species, dietary restriction is proven to be beneficial to health and life span. In primitive species, removal of gonadal germ cells has a similar positive effect on health and life span. These observations suggest a relationship between reproduction and health, but this relationship has been difficult to define, particularly in mammals. In female mammals, dietary restriction increases health span but also induces ovarian germ cell quiescence.A prominent example of the relationship between chronological life span, reproductive life span, and health is seen in human females. Chronological life span in humans has been extended dramatically over the last century. However, the timing of menopause or the end of the reproductive life span in human females has remained relatively constant over this same time period. Prior to reproductive decline, females hold a significant health advantage over males of the same age. However, at the time of reproductive decline, the increase of disease risks in women significantly outpaces those of men. This dependence on reproductive function for the maintenance of health is exemplified in women with premature ovarian failure, who suffer from a decline in health at a much younger age than in women with traditional menopausal timing.In primitive species, the reproduction-related effects on health span are thought to be due to the preservation of young, gonadal somatic cells which are protected from active germ cell signaling by removal of the germ cells. Previously, we replaced senescent ovaries of postreproductive female mice with young ovaries. The result of this manipulation was an increase in life span similar to that observed with dietary restriction . We alsoThe CBA/J strain (used in the current study) and the DBA strain of mice are unique in that they prematurely lose their ovarian follicles, becoming reproductively senescent by 10–12 months of age –6. A red ad libitum . Mice were maintained in an American Association for Accreditation of Laboratory Animal Care- (AAALAC-) approved facility in accordance with the National Institutes of Health animal-use guidelines. Animal care and use protocols were developed under National Research Council guidelines found in the Guide for the Care and Use of Laboratory Animals. This project was approved by the Utah State University Institutional Animal Care and Use Committee.Twenty-one-day- and eight-month-old CBA/J strain female mice were obtained from Jackson Laboratory . The 14-month-old female CBA/J mice were obtained from the National Institute on Aging rodent colony. All mice were housed in a standard laboratory animal environment . The mice were housed individually in ventilated cages  on corn cob bedding  changed once a week, with added enrichment, in a specific-pathogen-free colony where pathology on sentinel mice was done quarterly and pathological results showed no breach in this status. The mice received deionized water and a certified laboratory dietAnesthetics were used during surgery  and analgesia was provided for 48 hours postoperatively and longer if deemed necessary. Mice were euthanized by cervical dislocation. Immediately after cervical dislocation, thoracotomy was performed followed by rapid exsanguination via cardiocentesis. The heart and arterial tree were then removed from the mouse. Animals with acute but not severe weight loss were treated with subcutaneous fluids and moistened food. Animals with acute but not severe urine staining or rectal/vaginal prolapse were manually cleaned and treated with Desitin®. Mice were monitored at least twice daily and weights were recorded monthly, more frequently when concerns arose. Aged, moribund mice found with overt clinical signs (catatonia) were euthanized. Criteria for euthanasia specific for aged mice were determined in coordination with the attending veterinarian and included but were not limited to mice found in poor condition with or without crusting around the perineum and diarrhea, urine staining, persistent vaginal prolapse, chronic vulva/rectal swelling, kyphosis, respiratory distress, anorexia, poor coat condition and lack of grooming, moribund mentation, hind-limb weakness/paresis, wounds not healing, limited mobility, neoplastic growth, and unusual weight loss (or gain). Average weight loss in aged, female CBA/J mice from peak weight to death is approximately 12% per month . An incrAnimals were randomly assigned to control or ovarian transplantation groups as follows . n = 10).Six-month-old control mice: reproductively cycling mice were evaluated at six months of age ( n = 5).Thirteen-month-old control mice: acyclic mice were evaluated at 13 months of age ( n = 5).Sixteen-month-old control mice: acyclic mice were evaluated at 16 months of age (Controls consisted of the following. n = 5).Sixteen-month-old recipient mice: recipient animals remained intact but were acyclic at 12 months, at which time their senescent endogenous ovaries were removed and replaced with a pair of active donor ovaries from a 60-day-old mouse. These reproductively cycling mice were evaluated at 16 months of age ( n = 5).Twenty-one-month-old recipient mice: recipient animals received at 14 months were acyclic. At 17 months, their senescent endogenous ovaries were removed and replaced with a pair of active donor ovaries from a 60-day-old mouse. These reproductively cycling mice were evaluated at 25 months of age  and subsequent ovarian transplantation and received a pair of 60-day-old ovaries from a donor mouse of the same strain. Bilateral ovarian transplantation surgeries were performed as previously described . BrieflyData on vaginal cytology were collected for at least 10 consecutive days pre- and postoperatively to ensure (1) cessation of cycling at 12 months of age and (2) success of the ovarian transplantation procedure. Daily vaginal cytology was reinitiated beginning 10–14 days postoperatively. One estrous cycle was defined as the period from the day nucleated epithelial cells first appeared  to the day preceding the next appearance of nucleated epithelial cells in the vaginal smear, provided there was a period of leukocytic presence  in between. Estrus was determined by the presence of large, squamous epithelial cells, with or without nuclei. No immunosuppressive techniques were employed and no evidence of graft-versus-host disease was detected after transplantation or at death. Each female was housed individually.Mice that displayed cytological evidence of gonadal input prior to surgery at 12 or 17 months of age were excluded from analysis. Gonadal input was defined as cyclic changes on vaginal cytology, presumably due to cyclic influence of ovarian hormones. No gonadal input was defined as the lack of cyclic changes on vaginal cytology. Ovarian transplant recipients that failed to display evidence of gonadal input postoperatively based on vaginal cytology were also excluded from analysis. Gonadal input was determined by vaginal cytology analysis, as described in Surgical Procedures. Mice that displayed no cyclic activity on vaginal cytology for a 10-day period before and/or after surgery were determined to have no gonadal input for said period. Mice that displayed at least one full estrous cycle in a 10-day period before and/or after surgery were determined to have gonadal input for said period. Mice that fit these criteria were the only mice used for analysis throughout this study.Not all groups were subjected to each test of health span. Importantly, the 25-month-old group was often intolerant of metabolic or stressful manipulations and was included only in the magnetic resonance imaging (MRI) analysis procedures.The inverted cling grip test (ICGT) is a measure of overall strength and muscular endurance of the mouse . This tet0), and at 15, 30, 60, and 120 minutes after injection of 15% D-glucose (2.8 g/kg lean body mass). Calibration of the FreeStyle Freedom Lite Blood Glucose Monitoring System was performed using control test solutions provided by the manufacturer.An intraperitoneal (IP) glucose tolerance test (GTT) was performed in mice that were feed-deprived for 4-5 hours. We chose a 4-5-hour fast due to the metabolic differences between humans and mice and additionally because our aged females did not respond well to an overnight fast . Blood gt0). The sum of the trapezoidal areas between the 0-, 15-, 30-, 60-, and 120-minute time points corresponding to each animal was computed to obtain the AUC. The relative area values are reported as a percentage of the mean AUC of the six-month-old control cohort, which was defined as 1.0.Two drops of blood were collected, the first of which is discarded. The second drop was applied directly to a FreeStyle Freedom Lite test strip to obtain a blood glucose reading expressed as mg/dL. Glucose measurements at each time point were plotted and results were expressed as the area under the curve (AUC) for the 120-minute assay. The AUC corresponding to each animal was calculated by the trapezoid method , using aAlterations in mouse whole body composition were assessed using an EchoMRI-700 Magnetic Resonance Imaging system . The EchoMRI-700 system is housed in a dedicated area to minimize stress and the entry of disease or contaminants. Prior to each run, the system was calibrated using a standard provided by Echo Medical System. Each mouse was weighed and placed into an appropriate size tube. The tube was then placed in the EchoMRI-700 machine for measurements (~1 min). The output information included lean tissue mass, fat mass, free water, and total body water. No anesthesia was required for this procedure. post hoc test was used to determine difference between groups. Individual treatments were further analyzed by paired Student's t-test, two-tailed, unequal distribution of variance assumed. Test results were considered significant for P < 0.05.Statistical analysis was performed using GraphPad Prism 7.01 . A D'Agostino-Pearson omnibus test was used to determine normality. Data were analyzed with two-factor ANOVA and a Tukey-Kramern = 5) and tended to be reduced in 13-month-old mice , compared with six-month-old controls (n = 10). Sixteen-month-old recipient mice with young ovaries tended to fall sooner than the 16-month-old control mice .Performance on the ICGT defined as the average time (duration in seconds) before falling from the grid declined with age and with ovarian transplantation. The effect of age on overall muscle strength, as measured by the ICGT, is summarized in n = 5) and transplant recipient mice (n = 5) displayed no significant change in glucose tolerance, compared with young controls (n = 10).Because all groups did not have equivalent fasting glucose levels, glucose measurements at each time point were plotted and results were expressed as the area under the curve (AUC) for the 120-minute assay. AUC calculations for n = 5 and n = 5, resp.). Fat mass was reduced by 25% by ovarian transplantation in 16-month-old recipient mice (n = 5). The percentages of lean body mass and total body water were significantly reduced in 13-month-old control mice. The percentage of lean body mass and total body water in 16- and 25-month-old recipient mice (n = 5) was restored to the levels found in six-month-old control mice (n = 10) by ovarian transplantation.There were significant differences in body weight between age groups, but not due to treatment . Fat masSix-month-old female CBA/J mice are reproductively competent and have active ovaries that are reproductively cycling. By 12 months of age, ovaries in female CBA/J mice have become senescent and the mice have ceased reproductive cycling. In the current experiments, a group of 12-month-old mice received young ovaries from two-month-old donors. Therefore, the six-month-old control mice and 16-month-old recipient mice both possessed six-month-old ovaries and both benefitted from the effects of relatively young ovarian function. Because the 16-month-old cohorts of mice were collected prior to death, we were unable to collect life span data on these mice. However, previous studies using the same ovarian transplantation model in the same strain of female CBA/J mice resulted in a significantly increased life span in transplant recipients [Skeletal muscle function declines with age . BecauseIn our mice, a significant loss of grip strength occurred from six to 16 months of age. Ovarian transplantation moderately (16%) exacerbated this loss of grip strength in 16-month-old mice. This was unexpected as ovarian transplantation increased lean muscle mass by 8% in 16-month-old recipient females. Transplant recipients also weighed 5% less than 16-month-old control females. A decline in muscle mass (sarcopenia) is frequently observed in postmenopausal women . PreviouMice are inherently afraid of falling and motivation may have influenced the performance on this test. Studies in postmenopausal women found that mean anxiety/fear scores improved significantly with HRT , 23. OldGlucose intolerance increases with age. As a measure of glucose tolerance, we used a standard GTT assay. We used an IPGTT rather than an oral (PO) GTT as the PO test is associated with an increased level of stress on the tested subjects and the PO test is also associated with gastrointestinal variability in the timing and dose of glucose absorbed. Variability in the IPGTT is often attributed to the injection needle penetrating the intestines and glucose being variably deposited into the gastrointestinal tract rather than the intraperitoneal cavity. We utilized short-needled (6 mm) insulin syringes to minimize this possibility. Aged mice are often intolerant of insulin injections and thus our metabolic testing was limited to the glucose tolerance test in the current experiments.n = 4), but in 16-month-old mice with young ovaries only 60% displayed a monophasic glucose curve (n = 3) and 40% displayed a biphasic glucose curve (n = 2). Mice in all groups that displayed a biphasic glucose curve (n = 4) displayed lower fasting glucose levels, significantly greater body weight, heavier hearts and kidneys, lighter spleens, greater lean body mass, and lower fat mass and displayed increased grip strength, compared to mice with a monophasic curve (n = 21). The biphasic shape has been associated with normal glucose tolerance in humans and those with a monophasic plasma glucose curve have been shown to be at an increased risk for insulin resistance [n = 1) which were assumed to be healthier displayed a biphasic glucose curve. With the small numbers associated with these particular observations, any conclusions from these assessments are tenuous at best.The glucose AUC was not significantly influenced by age or ovarian transplantation. However, in 16-month-old controls, 80% of the mice displayed a monophasic glucose curve . Generally, total body water decreases with age, with the highest levels present at birth and a precipitous drop thereafter until death . Restoraα [Ovaries in our 13-month-old mice became senescent at about 10–12 months of age. Therefore, these 13-month-old mice have only been without active ovaries for approximately 1-2 months. It normally takes about 100 days for animals to respond/adjust to major changes in ovarian function . The “critical period” hypothesis suggests that there is an interval during perimenopause where hormone replacement therapy is effective, but efficacy decreases if therapy is initiated in the postmenopausal years , 28. Theα . In the α  and bothα . TherefoOverall, the restoration of health by transplantation of young ovaries appears to be relatively robust. A major limitation of this study was the modest number of animals available in each treatment group. This is not an unusual situation in aging studies. However, even with the small number of animals used, many tests displayed large differences between groups but often failed to reach statistical significance.In summary, we have shown that sarcopenia, which is negatively influenced by aging in females, can be positively influenced by reestablishment of active ovarian function in aged mice. However, much controversy remains regarding how the ovary provides the ovarian-dependent health advantage enjoyed by young females and its role in the restoration of health in aged females. These findings provide strong incentive for further investigation of the positive influence of young ovaries on restoration of health in postreproductive females.


Loss-to-follow-up (LTFU) throughout the Prevention of Mother-To-Child Transmission (PMTCT) cascade remains one of the major threats to the success of PMTCT programs. In this study, we aimed to determine the mother-to-child transmission rate in a programmatic setting and to determine factors associated with LTFU among enrolled mothers and unfavorable outcomes among HIV-exposed babies which includes being HIV positive, death and LTFU.A retrospective cohort study reviewing routinely collected data in an Integrated HIV care program, Mandalay, Myanmar in June 2016.LTFU means mother/infant missing appointed visit for more than three months.Of 678 pregnant women enrolled in PMTCT program between March 2011 and June 2014, one stillbirth and 607 live births were recorded in this cohort. Of 457 HIV-exposed babies with HIV-test recorded at the end of the intervention, nine (2%) were HIV-positive. Pregnant women’s and exposed-babies’ LTFU rate was 7 per 1000 person-years, and 10 per 1000 person-years respectively. PMTCT option B protocol was found to be significantly associate with maternal LTFU [adjusted Hazard Ratio (aHR) 95% CI: 3.52 (1.38–8.96)] when compare to mothers receiving option B+/lifelong antiretroviral therapy (ART). Weight <2.5 Kg at enrolment, receiving mixed-feeding, vaginal delivery and option B PMTCT protocol were significantly associated with unfavorable outcomes among exposed babies .Mother-to-child HIV transmission rate in this public hospital-based program was lower than the 5% national target, which indicates a successful PMTCT intervention. However, a high proportion of HIV-infected mothers and exposed babies LTFU was recorded. Lifelong ART provision to HIV-positive pregnant women was shown to reduce exposed babies’ LTFU, death and transmission rate (unfavorable outcomes) in this setting. Lessons learned from this program could be used to inform policy and practice in the country, while the programmatic challenge of LTFU should be urgently addressed. Mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) remains a major health problem worldwide even though the rate of transmission globally fell from 26% in 2009 to 16% in 2013 and 10% in 2015[In 2013, 54% of pregnant women in low and middle-income countries did not receive routine HIV testing during antenatal care (ANC), which is a key step to accessing HIV prevention, treatment and care. In 2015In Myanmar, HIV prevalence in the adult population aged 15 years and older was estimated at 0.54% in 2014; it has considerably declined from 0.94% in 2000,13. The The PMTCT program in Myanmar is routinely monitored and evaluated however there has been no in-depth analysis of outcomes and factors associated with successful prevention, neither has been documented on lessons learned that could inform policy and practice.The aim of this study was to determine the mother-to-child transmission rate among HIV-infected mothers and their exposed babies enrolled in the Mandalay Central Women Hospital PMTCT program between March 2011 and June 2014 and to investigate the factors associated with LTFU among enrolled mothers as well as factors associated with unfavorable outcomes among HIV-exposed babies.Permission for the study was obtained from National AIDS Program, Myanmar. Ethics approval was obtained from the Ethics Review Committee at Department of Medical Research, Yangon, Myanmar and the International Union Against Tuberculosis and Lung Disease (The Union) Ethics Advisory Group, Paris, France.This was a retrospective cohort study using routinely collected data on PMTCT of an Integrated HIV Care (IHC) Program in Myanmar.Myanmar is Southeast Asian country bordering India, Bangladesh, China, Laos and Thailand. The population of Myanmar was 53,259,000 in 2013 with 33% of the population living in urban areas. About 6 st session- at enrolment visit, 2nd during the blood collection day, and 3rd at a clinic appointed day) by peer counsellors and medical social workers from the hospital. The program collaborates with an extensive PLHIV network which provides comprehensive counselling services and LTFU tracing. The Union supports formula feeding to exposed babies until the 9th month of age and provides counselling and demonstration on formula feeding preparation in collaboration with the medical saffs from the hospital. Last, all enrolled mothers and babies are followed at the CWH PMTCT clinic until 18th month post-partum or until the child HIV status have been confirmed. Early infant diagnosis (EID) is also provided to HIV exposed infants.Since 2011, The Union has been implementing PMTCT services in Central Women Hospital in Mandalay as a PMTCT clinic under IHC program, in collaboration with the National AIDS Program (NAP) and public hospitals and clinics under the Ministry of Health and Sports (MoHS). The PMTCT clinic enrolled HIV-infected pregnant women as well as post-partum women along with their exposed babies mainly delivered at CWH also other public hospitals and private clinic, and provided comprehensive PMTCT services. This integrated PMTCT care is delivered by obstetricians and pediatricians from CWH and physicians from Mandalay General Hospital as well as medical officers employed by the Union. Three counselling sessions on adherence to ART and regular follow up are offered to PMTCT women (13 before 2015 and 500 cells/mm3 after 2015)[HIV-infected pregnant women who were eligible for treatment were offered antiretroviral therapy (ART) for life if their CD4-counts is lower than cutoff point (CD4-count cutoff was 350 cells/mmPMTCT option A (prior to 2013): women received AZT onlyPMTCT option B (from 2011 to 2014): women received triple ART, during pregnancy, delivery and discontinued one week after breast feeding was stoppedPMTCT option B+ (2014 to date): women received triple ART during pregnancy, delivery and continued for life.At enrollment, every mother and child are given a unique registration code. Baseline demographic characteristics along with history of pregnancy and delivery are collected and CD4-count for the mother is tested. After delivery, mother and infant are followed-up until 18 months post-partum. The follow-up visit frequency varied depending upon the attending physician, feeding practices, the health status of the mother and the infant and distance of patients’ resident. In every follow-up visit, mothers are asked to bring along their infants. Infants receive HIV virological testing (DNA/RNA PCR essay) at the age of 4–6 weeks and 9 months, and are tested for HIV serology by using rapid tests (Determine HIV-1/2) at the age of 9 and 18 months. The indeterminate results at 9 months are confirmed with DNA/RNA assay between 9 and 18 months of age. HIV-positive infants were transferred out to Pediatric IHC clinic for ART initiation of ART and continuation of care. HIV-infected mothers and their exposed infants enrolled in the PMTCT program and received PMTCT intervention in Central Women Hospital, Mandalay, Myanmar between March 2011 and June 2014.In every visit at the PMTCT clinic, medical doctors filled out the standardized PMTCT visit forms, and trained data staffs transcribed the paper-based data into the electronic database of IHC program after each clinic session.Collected variables included: PMTCT protocol, CD4-count before delivery, baseline WHO staging, mode of delivery, place of delivery, pregnancy outcomes, employment status and mother literacy status, sex of exposed infants, infant weight at enrolment, and mode of feeding.Data were extracted from the electronic database of the IHC program and imported into STATA version 14.2 . Data were anonymzed and de-identified prior to analysis.th June 2016 for all exposed babies to have follow-up period up to 18 months of age. Cox proportional hazards models were used to determine the factors associated with mothers’ and exposed babies’ outcomes. P-value <0.05 was regarded as significant. A set of a priori variables based on knowledge from previous literatures such as PMTCT protocol, CD4 before delivery and mode of delivery were included as covariate in multivariable analysis to find the association with LTFU outcome of HIV-infected mothers. Weight at enrollment, mode of feeding, maternal PMTCT protocol, duration of ART and mode of delivery were used to find the association with unfavorable outcomes among exposed infants. [The number and proportion of mother LTFU, HIV transmission and unfavorable outcome of exposed babies  were calculated. Rate of LTFU among mothers and of unfavorable outcomes among exposed babies were calculated with censoring on 30th June 2016. Out of 607 HIV-exposed infants, 99 (16%) were lost to follow-up before the final diagnosis was made with LTFU rate of 10 per 1000 person-years (95% CI 8.52–12.9). Of 457 babies with HIV-test recorded 9 (2%) were tested HIV-positive, as shown in A total of 678 pregnant women were enrolled into the PMTCT program between March 2011 and June 2014. The median age (interquartile-range) at enrollment was 29 (26–33) years. Among these women, 19 (2.8%) were lost to follow-up before delivery, 1 (0.2%) had stillbirth and the remaining 607 (99.8%) live births. The LTFU rate among mother was 7 per 1000 person-years (PY) (95% CI 6–9). Among women who had delivery history (608), 12(2%) were death, 81 (13%) were LTFU, 497 (82%) were transferred out to other IHC sites after delivery and 18 (3%) were on regular follow-up by 30This is the first study looking at the outcomes of HIV-infected mothers and their exposed infants enrolled in an integrated, hospital-based PMTCT program, in Myanmar. With a recorded mother-to-child HIV transmission risk of less than 2%, considerably lower than the 5% target set by the National AIDS Program, Myanmar. So this program could be considered successful at the national and regional level . Howeverth days after delivery which may or may not be the same as PMTCT clinic appointment. Therefore, this is the possible reason that mother did not attend PMTCT clinic after delivery. In addition, most working women return to work soon after delivery, making regular PMTCT follow-up challenging and competing with other life priorities.The proportion of mothers who were LTFU after delivery was found to be five times higher than LTFU before delivery. There are many reasons of higher LTFU after delivery. The pregnant women have to attend ante-natal care clinics (ANC) regularly and therefore have less chance of dropping out from the PMTCT clinic during AN period which in this program is integrated into ANC and co-located in the same compound. HoweverThe LTFU rate in PMTCT IHC cohort was higher than adult IHC program cohort.This canIn our study, pregnant women who were not on ART or were on option B PMTCT protocol were more likely to be LTFU compared to women on option B+ protocol (lifelong ART). Several different factors such as having later gestational age at ART initiation, type of PMTCT protocol (single does NVP), being newly diagnosed with HIV in the current pregnancy, stigma and discrimination, low knowledge of PMTCT program, low coverage of PMTCT programs, longer waiting time and provider clinical knowledge and attitude were associated with LTFU ,21,24–26Exposed babies with body weigh less than 2.5 kg were more likely to experience unfavorable outcomes in this setting. This low weight at enrolment is associated with in utero transmission and pre-term delivery. In addition, vaginal delivery and mixed infant feeding were found significantly associated with unfavorable outcomes among exposed babies in this cohort; these factors were previously shown to be associated with such outcomes in other studies,18,27. EThis study has several strengths. First, the routinely collected program data was used which reflect the real situation in the field; therefore the findings could give reasonable and programmatic feedback to the program. Secondly, the sample size in this study was relatively large as well as the completeness and robustness of the data compared to other PMTCT program descriptions in other countries,28–30. LThe study did not come without certain limitations. Since this study used routinely collected data from an ongoing program, a certain amount of missing values were found and excluded from the analysis. Further strengthening of the monitoring and evaluation and reporting and recording systems is recommended. The reasons behind the LTFU of this programmatic cohort have not been explored in this study; further quantitative and qualitative studies were planned to conduct in order to address this most challenging programmatic outcome. Furthermore, the linkage between the antenatal HIV testing facility and PMTCT services were not looked at in this study where Lost to follow up was occurred up to 49% in low and middle income countries . Lastly,A low mother-to-child HIV transmission rate has been recorded in a public hospital-based program in Mandalay, Myanmar, which indicates a successful PMTCT intervention. There are several factors currently adopted in this setting such as integrated PMTCT care, counselling, LTFU tracing, provision of formula feeding and one stop services for both mothers and babies until confirmed HIV diagnosed of exposed babies, might have contributed to the overall success of this intervention, as evidenced by the low mother to child HIV transmission rate reported. According to the evidence produced by this study, we recommend that elements of the comprehensive package of this PMTCT program might be considered to be adopted by the National AIDS Program and expanded to other part of the country. However, high LTFU of HIV-infected mothers and exposed babies was also recorded. Lessons learned from this program could be used to inform policy and practice in the whole country, while the programmatic challenge of LTFU should be urgently addressed.S1 Table* , HR, hazard ratio; ¥AZT, Zidovudine; €NVP, Neviriapine; δ VD, Normal vaginal delivery; λ LSCS, Lower segment caesarean session.(DOCX)Click here for additional data file.S1 File(DTA)Click here for additional data file.


Despite robust empirical support for the efficacy of trauma-focused treatments, the dissemination proves difficult, especially in relation to patients with comorbid psychosis. Many therapists endorse negative beliefs about the credibility, burden, and harm of such treatment.This feasibility study explores the impact of specialized training on therapists’ beliefs about trauma-focused treatment within a randomized controlled trial.n=16) credibility, expected burden, and harm expectancies of trauma-focused treatment were assessed at baseline, post-theoretical training, post-technical training, post-supervised practical training, and at 2-year follow-up. Credibility and burden beliefs of therapists concerning the treatment of every specific patient in the trial were also assessed.Therapist-rated (n=79), pre-treatment symptom severity was not associated with therapist-rated credibility or expected burden of that specific treatment. Treatment outcome had no influence on patient-specific credibility or burden expectancies of therapists.Over time, therapist-rated credibility of trauma-focused treatment showed a significant increase, whereas therapists’ expected burden and harm expectancies decreased significantly. In treating posttraumatic stress disorder (PTSD) in patients with psychotic disorders (These findings support the notion that specialized training, including practical training with supervision, has long-term positive effects on therapists’ credibility, burden, and harm beliefs concerning trauma-focused treatment.Specialized training improved therapists’ credibility, burden, and harm beliefs.Patients’ symptom severity and treatment outcome did not affect these beliefs.Replication of our findings in larger cohorts with a control group is warranted. Because there is strong empirical support for the efficacy of trauma-focused treatments such as prolonged exposure therapy (PE), eye movement desensitization and reprocessing therapy (EMDR), and cognitive therapy  found that only 6.3% of the patients received at least one session of evidence-based trauma-focused treatment during the first six months of their treatment  and patient factors , therapist characteristics and, more specifically, therapists’ beliefs about trauma-focused treatments appear to be an important cause of underutilization of evidence-based interventions for PTSD  are of little value to their clinical practice  beliefs about the possible negative consequences of using trauma-focused treatments for their patients. The most important harm expectancy of therapists is that trauma-focused treatment will destabilize the patient and exacerbate symptoms, which could result in various adverse events, for example, crises, suicide attempts, hospitalization, revictimization, and dropout  and emotional appeals  and by using exercises that prompt both explicit and implicit learning  that found both PE and EMDR to be effective and safe in participants diagnosed with both a PTSD and a psychotic disorder  that received either PE or EMDR treatment during the trial from one of the 16 participating therapists.The inclusion criteria for this feasibility study were a) no previous training in PE or EMDR and b) consent to participate in monthly expert supervision sessions during the trial. The participants were 16 therapists  working at 13 mental healthcare organizations in the Netherlands. Twelve therapists were female and four were male. Their mean age was 37.1 (7.59) years, and on average, they had been working as a therapist for 8.6 (7.6) years. All therapists worked mainly with patients with psychotic disorders, were specialized in cognitive behavior therapy for psychosis, had no previous experience in trauma-focused treatment, and volunteered to participate in a trial for trauma-focused treatments in psychosis. To test the influence of symptom severity and treatment outcome on therapists’ beliefs, we included patients . Therapists responded on a visual analog scale (VAS) ranging from 0 (“disagree strongly”) to 10 (“agree strongly”) with a higher score representing a higher level of credibility of trauma-focused treatment. The therapists’ credibility assessment was inspired by the Credibility/Expectancy Questionnaire, a short measure of patient-rated credibility of treatment that has shown high internal consistency and good test–retest reliability  to 10 (“agree strongly”). Two statements concerned burden expectancies  and five statements concerned harm expectancies of trauma-focused treatment . These statements were inspired by the Distress/Endorsement Validations Scale  with three statements . Again participating therapists responded on VAS (0–10). The ratings concerned the therapist's beliefs about the treatment for that specific client.Patient-specific burden expectancy of trauma-focused treatment was assessed in the same way with two statements on a VAS .pre-treatment symptom severity for the specific patients  was established with the Auditory Hallucination Rating Scale , post-theoretical training, post-technical training, post-practical training, and at 2-year follow-up. Experts in the target treatments provided a 4-day training in PE (AVM and RDK) and a 4-day training in EMDR (ADJ). All therapists attended both trainings and all delivered both therapies during the trial. Standard protocols of these two guideline trauma-focused treatments were used  days of the PE and EMDR trainings were mainly theoretical, consisting of the following elements: (1) theoretical principles; (2) efficacy and safety of the treatment ; (3) treatment rationale and procedures; (4) specific techniques and skills; and (5) practicing techniques and skills in role play with peers.The third and fourth days of the two trainings were technical and were spread over a 5-month period. During this phase, the participants had to treat at least two patients with PE and two with EMDR. These sessions were videotaped, viewed, and (plenary) discussed focusing on the technical aspects of conducting the therapies.Recruitment for the trial ran from September 2011 through April 2013. During this training phase, therapists treated patients and underwent monthly 4-h group supervision sessions (group size 6–8) that were led by experts (2 h by AVM in PE and 2 h by CDR or ADJ in EMDR). In these supervision sessions, video recordings of complicated treatment sessions were viewed and (plenary) discussed.At the end of the trial, the therapists resumed their regular function in clinical practice, mainly in cognitive behavior therapy for psychosis. Two years after the end of the trial, the participating therapists were surveyed about their use of PE and EMDR. The questions were “Are you still using trauma-focused treatments in patients with psychosis?”; “How many patients with a psychotic disorder did you treat with trauma-focused treatment since the closure of the trial?”; “In case you did not treat any patients with trauma-focused treatment, why not?”; and “What factor influenced your daily practice most ?”n=39) or EMDR (n=40). All sessions were videorecorded. Session one comprised psycho-education  and the development of a hierarchy of the worst (and most re-experienced) trauma memories. No trauma-focused treatment was provided in this first session. Therapists rated their credibility and expected burden of the treatment of every specific patient at pre-treatment (after session 1) and at post-treatment (after the last therapy session). For each patient, the independent assessors assessed pre-treatment symptom severity and post-treatment severity of PTSD symptoms.During the supervised practical training phase, the therapists treated patients included in the trial. In this study, the 79 participating patients were randomly assigned to receive eight weekly 90-min sessions of either PE ; and(4) there were no differences in efficacy . In this study, we pooled the data on therapists’ beliefs of the trauma-focused treatments PE and EMDR because (1) the PE and EMDR trainings took place in the same period; (2) all therapists provided both treatments; (3) there was no difference in treatment allegiance of therapists to PE or EMDR  were used to investigate effects between the different time points, that is, during theoretical training, technical training, practical training, and the follow-up period.Influence of symptom severity on patient-specific credibility and burden of trauma-focused treatment. We computed bivariate Pearson product–moment coefficients between the dependent therapist-rated variables “pre-treatment credibility of treatment” and “pre-treatment expected burden of treatment” and five independent variables representing pre-treatment symptom severity . Since these data are nested within therapists, we used linear mixed models with a correction for therapist level  to test these associations. The independent variables were entered (forced simultaneous entry) into two separate linear mixed models analyses (one for credibility and one for expected burden) to preserve degrees of freedom  were used to analyze changes in the severity of PTSD symptoms and in therapist-rated credibility and burden of trauma-focused treatment, between pre-treatment and post-treatment. Then, bivariate Pearson product–moment coefficients were computed between change scores of PTSD and change in therapist-rated credibility and burden. We performed linear mixed models (intention-to-treat) with a correction for therapist level to test the relationship between the dependent variables “change in credibility” and “change in expected burden” and the independent variable “change in PTSD symptom severity.”Three participating therapists missed the post-practical training assessment and two missed the 2-year follow-up assessment due to pregnancy leave or prolonged illness (unrelated to work as clinician).F=11.75, p<0.001). Credibility increased significantly during the theoretical training =3.36, p=0.002, 95% CI ). This effect was sustained during the subsequent phases with small non-significant increases in credibility in every training phase. Therapist-rated burden expectancies showed a significant decrease over time =9.20, p<0.001). Burden expectancies decreased significantly after theoretical training . This was followed by a small, non-significant increase  during technical training. Thereafter, the level of expected burden was relatively stable, showing no significant changes during the subsequent phases. Therapist-rated harm expectancies of trauma-focused treatment significantly decreased over time =4.44, p=0.005). After an initial small, non-significant decrease after theoretical training , harm expectancies showed a significant, but limited, increase during technical training  and then a limited (border) significant decrease during the practical training phase .n=14) showed that 12 participating therapists (85.7%) still used trauma-focused treatments (PE or EMDR) in patients with psychotic disorders. In the last 2 years, the therapists had treated (on average) 12.3 (8.1) patients. The two participants that were no longer using trauma-focused treatments in psychosis had changed their job and were no longer working with this patient group. The majority of the participating therapists (78.6%) indicated that the supervised practical training had most strongly impacted their clinical behaviorThe 2-year follow-up survey , the results of the paired-samples t-tests showed no significant change in either therapist-rated credibility  or burden expectancies  during treatment  or burden . The mixed model analyses with a correction for therapist level also showed results far from significance.Of the 79 patients treated by the 16 therapists, 8 (10.1%) did not attend the post-treatment assessment of severity of PTSD symptoms. Also, 14 patients (17.7%) had no post-treatment therapist ratings of credibility and expected burden, either because the patient dropped out of treatment or because the therapist became ill. For 63 patients (79.7%), all data were present. Although the mean CAPS severity score for these patients showed a significant change during treatment  as sensitivity analyses, and all yielded results similar to the original analyses.All the analyses in this section were repeated with the last observation carried forward  and post-training telephone consultation (moderate effect) to increase credibility of PE  rebound during the technical training when therapists started to treat patients. This partial recovery of burden expectancies and the fact that these showed no further significant decrease during the practical training phase or follow-up may be explained by the fact that a certain level of burden of trauma-focused treatment is probably realistic—especially when considering that the therapists  were novices to trauma-focused treatment and immediately started treating a patient group characterized by severe PTSD, many comorbidities, and severe childhood traumas , the characteristics of specific patients  and treatment outcome had no influence on therapists’ credibility and burden beliefs concerning trauma-focused treatment. The only significant association was a positive correlation between pre-treatment PTSD symptom severity and pre-treatment credibility of trauma-focused treatment which, in the multiple regression analysis, was lost after correction for the variability explained by the other pre-treatment patient characteristics. This positive correlation tentatively suggests that therapists, with high mean scores of credibility of trauma-focused treatment at that time, may have reasoned that with severe PTSD trauma-focused treatment was probably going to be effective. Greater severity of pre-treatment PTSD was indeed found to be related to a greater reduction in PTSD symptoms during treatment in participants with severe mental illness  are the fact that participating therapists were not randomly selected, the lack of a control group, and the small sample size. A strength of this contextual factor is that fidelity to the protocol was high; however, this also limits the generalizability. Moreover, participating therapists voluntarily participated in a trial on trauma-focused treatment in psychosis, despite (at that time) limited empirical evidence regarding its efficacy and safety. This may have resulted in ceiling and floor effects. Furthermore, participating therapists concurrently received PE and EMDR training, which might have had a differential influence on their beliefs. The data for these two trauma-focused treatments were pooled as we could not isolate carry-over effects. Unfortunately, at the patient level we did not assess harm expectancies. Also, patient-specific burden expectancies (rated by the therapists) only concerned therapist-rated burden to the therapist and not to the patient. Future studies could include these latter assessments. Finally, we used non-validated measures to assess therapist's beliefs, although internal consistency scores were satisfactory.In conclusion, the present findings support the notion that specialized trauma-focused treatment training, including acquisition of experience, increases credibility and reduces beliefs about burden and harm. This is underlined by the finding that the effects were sustained on the long term and were unaffected by specific patient characteristics and treatment outcome. Future studies could use a similar design with larger samples of frontline therapists in clinical practice. These studies should include a control group and may test whether the level of experience influences training effects. These studies could also compare theoretical and technical training with training that is augmented with an expert supervision trajectory. It is important to establish what beliefs are most strongly related to long-term clinical behavior and what elements of training have the strongest influence on these beliefs (Ruzek et al.,


This study examined the predictors of loss to follow-up in long-term supportive periodontal therapy in patients with chronic periodontitis.A total of 280 patients with moderate to severe chronic periodontitis in a tertiary care hospital in China were investigated and followed over the course of study. Questionnaires on clinical and demographic characteristics, self-efficacy for oral self-care and dental fear at baseline were completed. Participants were followed to determine whether they could adhere to long-term supportive periodontal therapy. Binary logistic regression analysis was used to examine the association between clinical and demographic characteristics, self-efficacy for oral self-care, dental fear and loss to follow-up in long-term supportive periodontal therapy.p = 0.006], severe periodontitis , periodontal surgery , and middle and low-scoring of self-efficacy scale for self-care groups. The adjusted ORs of loss to follow-up for the middle- (54–59) and low-scoring groups (15–53) were 71.899  and 4.800 , respectively, compared with the high-scoring SESS group (60–75).The loss to follow-up in long-term supportive periodontal therapy was significantly associated with age [adjusted OR = 1.042, 95% confidence interval (CI): 1.012–1.074, Age, severity of periodontitis, periodontal surgery and the level of self-efficacy for self-care may be effective predictors of loss to follow-up in long-term supportive periodontal therapy in patients with chronic periodontitis. Chronic periodontitis was the primary cause for adult tooth extraction in a report from the third national oral epidemiology investigation in China. ChronicInitial periodontal therapy effectively removes bacterial plaque and relieves symptoms. However, long-term periodontal health maintenance largely depends on regular and frequent check-ups. Therefore, whether patients enter into supportive periodontal therapy and adhere to long-term follow-ups after completion of the initial therapy is closely related to the clinical outcome of periodontitis. Unfortunately, a considerable number of patients are lost during long-term supportive periodontal therapy in clinical practice despite dentists repeated emphasis on the importance and necessity of follow-up in long-term supportive periodontal therapy and encouragement to enter supportive periodontal therapy and insistence on regular follow-ups.Bandura found that self-efficacy, as a core concept of social cognitive theory, played a key role in individual action-taking and emotion responding. Self-efApplication of self-efficacy theory in the oral hygiene field was reported in several studies. Earlier evidence demonstrated positive associations between self-efficacy and improved clinical indexes and self-care behaviors, such as brushing and flossing frequency–15. OralChina is the most populous developing country in the world. The third national oral health epidemiological survey in China reported 38.9% and 71.3% of patients with ≥4 mm clinical attachment loss (CAL) in age brackets 35–44 and 65–74 years old, respectively. The curA prospective cohort study was performed from November 2014 to December 2016. Ethical approval was obtained from the Tianjin Medical University Ethics Committee prior to the start of the study (No. TMUHMEC2014001).Participants retained the right to decide their continued participation in this study at all times. The researchers promised that all information related to the participants would not be disclosed to ensure the safety of participants’ privacy. All patients provided informed consent prior to participation.This study recruited 280 subjects with moderate to severe chronic periodontitis at a tertiary grade hospital of stomatology between November 2014 and January 2015 in China. Sample size requirements were determined using logistic regression. The sample size should be 5–10 times the number of independent variables, and our study included 21 independent variables. The calculated sample size ranged from 126 to 252 subjects considering a loss to follow up rate of 20%. Therefore, we recruited 280 subjects into the study.Periodontitis was classified according to the American Academy of Periodontology (AAP) and the Centers for Disease Control and Prevention (CDC). Mild periodontitis was defined as ≥2 interproximal sites with CAL ≥3 mm (not on the same tooth) and ≥2 interproximal sites with PD ≥4 mm (not on the same tooth) or ≥1 interproximal site with PD ≥5 mm. Moderate periodontitis was defined as ≥2 interproximal sites with CAL ≥4 mm (not on the same tooth) or ≥2 interproximal sites with PD ≥5 mm (not on the same tooth). Severe periodontitis was defined as ≥2 interproximal sites CAL ≥6 (not on the same tooth) and ≥1 interproximal sites with PD ≥5 mm.Patients were excluded if they exhibited physical limitations influencing manual dexterity; had diabetes mellitus, immunodeficiency, or fewer than 20 teeth; or were taking medications that affect the development of inflammation of periodontal tissues. Patients who required prophylactic antibiotic premedication, had undergone extensive non-surgical periodontal treatment within the previous 6 months and periodontal surgery within the previous 2 years or were undergoing any active periodontal treatment were also excluded,20.Participants were classified into two groups according to the outcome loss to follow-up in long-term supportive periodontal therapy. Group 1 included 160 patients who were lost during follow-up periodontal treatment. These patients completed the initial therapy but failed to enter supportive periodontal therapy or entered into supportive periodontal therapy but were lost in long-term periodontal therapy. Group 2 included 120 patients who received periodontal therapy for over 2 years.An investigator-training meeting was held prior to the start of the study to confirm the entire research process and unify operative standards. Investigators first used a uniform description to explain the purpose and significance of the study to ensure that all of the subjects provided informed consent to participate in the research. Investigators provided concise instructions on the method and announcements before the patients independently completed the questionnaire. Investigators immediately clarified their explanations if the patient had concerns or questions to ensure that all patients understand and completed the questionnaire fully and correctly. Two investigators immediately confirmed the completeness and validity of the questionnaire to ensure its validity and eligibility.Clinical parameters were obtained from periodontal examinations performed by one dentist at baseline. This clinician was 30 years old with an MD in stomatology and more than 6 years of clinical periodontitis diagnosis and treatment experience. All patients were given tooth-brushing instructions that included the Bass method and instPatients’ demographic data and clinical parameters, probing depth, clinical attachment loss, the distance from gingival margin to cementoenamel junction and numbers of teeth present were recorded during the initial examination at baseline. One dentist measured probing depth, clinical attachment loss and the distance from gingival margin to cementoenamel junction at six sites per tooth using a Williams probe that rounded to the nearest millimeter. Probing depth was the distance from the gingival margin to the bottom of the pocket. Clinical attachment loss was the distance from the bottom of the pocket to the cementoenamel junction. We also measured furcation involvement using a Glickman index value but did not incorporate this measurement into our study.p<0.001) in Japan.Kakudate et al. developed a task-specific SESS and used it in patients with periodontal disease. SESS is composed of three subscales: self-efficacy for dentist consultations , self-efficacy for brushing teeth  and self-efficacy for dietary habits ,22. A fiDi Wu et al. (2013) translated SESS into Chinese, amended it to consider the cultural background in China, and re-translated it into English to ensure a valid translation. The revLoss to follow-up was defined if the patient did not present him- or herself at an appointment and did not express a desire to receive consultation within 1 month from the day of the appointment.Statistical analyses were performed using SPSS statistical software . The Kolmogorov-Smirnov test was used to verify the normality of all variables. The chi-square test was used to examine the statistical significance of gender, educational level, monthly family income, tooth number group, severity of periodontitis, periodontal surgery, and the level of self-efficacy for self-care between the two groups. Independent sample t test was used to detect the statistical significance of age, probing depth, clinical attachment loss and SESS scores between the two groups. The Mann-Whitney U test was used to analyze differences in dental fear scale score, self-efficacy for dentist consultations subscale score, self-efficacy for brushing the teeth subscale score, and self-efficacy for dietary habits subscale score between the two groups. Binary logistic regression analysis was used to assess associations between clinical and demographic characteristics, self-efficacy for self-care and patient’s loss to follow-up in long-term supportive periodontal therapy. Variables that exhibited a significant association between group 1 and group 2 were used as independent variables, and loss to follow-up in long-term supportive periodontal therapy was the dependent variable. The results are presented as crude and adjusted ORs with 95%CIs. P-values <0.05 indicated significance.Age, clinical attachment loss and SESS were normally distributed. Probing depth, DFS score, self-efficacy for dentist consultations subscale score, self-efficacy for brushing the teeth subscale score, and self-efficacy for dietary habits subscale score exhibited a skewed distribution.A total of 160 men and 120 women were recruited into the study. A total of 160 subjects failed to enter into supportive periodontal therapy (Group 1), and 120 participants attended regular check-ups and entered into supportive periodontal therapy (Group 2).p = 0.037). The educational level of the patients in group 2 was higher than group 1 (p = 0.032). The severity of periodontal disease of the patients in group 2 was more serious than the patients in group 1 (p = 0.001). More patients in group 2 underwent with periodontal surgery than group 2 (p = 0.001). No significant associations in gender, monthly family income, number of teeth present, probing depth, clinical attachment loss, or DFS score were observed in either group.Tertile cutoff points for the low-, middle-, and high-scoring SESS groups were 15–53, 54–59, and 60–75, respectively. The mean SESS score and standard deviation (SD) of all participants were 57.03±9.08. The mean SESS score and SD of group 1 was 52.69±8.04, which indicated a middle-scoring SESS group. The mean SESS score and SD of group 2 was 62.83±6.91, which indicated a high-scoring SESS group.p<0.001), SESS score group (p<0.001), self-efficacy for dentist consultations subscale score (p<0.001), self-efficacy for brushing the teeth subscale score (p<0.001), and self-efficacy for dietary habits subscale score (p<0.001) significantly correlated with loss to follow-up.This study revealed significant difference between China and developed countries in the proportion of patients entering into supportive periodontal therapy. Several studies in developed countries reported a higher proportion of patients entering into supportive periodontal therapy than the proportion who fail to enter into supportive periodontal therapy in long-term periodontal therapy,16,24,25A higher level of oral hygiene-related self-efficacy was found in Germanic patients with periodontal disease. Our stuWoelber demonstrated that oral hygiene-related self-efficacy was a key factor in oral health behavior and effectively predicted patient oral health outcome in Germany. KakudatWe observed that in China, older patients were more likely to fail to enter into supportive periodontal therapy than younger patients were, which is inconsistent with the previous studies that found that patients who were older tended to remain in periodontal treatment,16,31,32No significant relationship was observed between compliance and gender, which is consistent with earlier reports–37. The There are several limitations to this study that should be acknowledged. First, numerous variables are used to diagnosis periodontitis, and theThese findings suggest that age, periodontitis severity, periodontal surgery and the level of self-efficacy for self-care may be effective predictors of loss to follow-up in long-term supportive periodontal therapy in patients with moderate to severe chronic periodontitis. Older patients with more severe periodontitis and lower self-efficacy for self-care were more easily lost. The screening of patients who tend to become lost in long-term supportive periodontal therapy during an early visit would allow the dental staff to provide targeted and effective support to reduce the number of patients lost to follow-up in long-term periodontal treatment and promote the periodontal health of the patients with periodontitis.S1 File(ZIP)Click here for additional data file.


Current studies of depression among people living with HIV focus on describing its point prevalence. Given the fluctuating nature of depression and its profound impacts on clinical and quality-of-life outcomes, this study aimed to examine the prevalence, recurrence and incidence of current depressive symptoms and its underlying catalysts longitudinally and systematically among these individuals.We conducted a prospective cohort study between October 1, 2007 and December 31, 2012 using longitudinal linked data sources. Current depressive symptoms was identified using the Centre for Epidemiologic Studies Depression Scale or the Kessler Psychological Distress Scale, first at baseline and again during follow-up interviews. Multivariable regressions were used to characterize the three outcomes.Of the 3,816 HIV-positive participants, the point prevalence of depressive symptoms was estimated at 28%. Of the 957 participants who were identified with depressive symptoms at baseline and who had at least two years of follow-up, 43% had a recurrent episode. The cumulative incidence among 1,745 previously depressive symptoms free participants (at or prior to baseline) was 14%. During the five-year follow-up, our multivariable models showed that participants with greater risk of recurrent cases were more likely to feel worried about their housing situation. Participants at risk of developing incident cases were also likely to be younger, gay or bisexual, and unable to afford housing-related expenses.Depressive symptoms are prevalent and likely to recur among people living with HIV. Our results support the direction of Ontario’s HIV/AIDS Strategy to 2026, which addresses medical concerns associated with HIV (such as depression) and the social drivers of health in order to enhance the overall well-being of people living with or at risk of HIV. Our findings reinforce the importance of providing effective mental health care and demonstrate the need for long-term support and routine management of depression, particularly for individuals at high risk. Depression affects up to half of people living with HIV, a prevalence that is two to four times higher than that found in the general population . Over 50Growing evidence supports a bi-directional relationship between HIV and depression involving a number of biological, psychosocial and social factors ,14–16. TTo date, most studies about the prevalence of depression among people living with HIV have used cross-sectional designs ,15. Six Given the fluctuating nature of depression over the life span and its profound impacts on clinical and quality-of-life outcomes, our study aimed to examine the prevalence, recurrence, and incidence of current depressive symptoms longitudinally and systematically among people living with HIV. We also characterized these three outcomes by HIV-positive participants’ socio-demographic characteristics, housing and neighbourhood conditions, substance-use behaviours and health status over a five-year follow-up period. Understanding change in the burden of depressive symptoms and the underlying catalysts of the condition from a longitudinal perspective would be important to program planners, policy-makers, and health care providers when planning and implementing effective mental-health programs and interventions for people living with HIV.We conducted a prospective cohort study between October 1, 2007 and December 31, 2012 by linking unique encoded identifiers from the Ontario HIV Treatment Network Cohort Study (OCS) with national and provincial administrative health databases held at the Institute for Clinical Evaluative Sciences (ICES). Details about the linked data source have been provided in a recent study . We haveThe OCS is a multi-site HIV cohort, described in detail in a previous publication . HIV-posWe used four administrative health databases to obtain additional information about participants’ past diagnoses of depression, other psychiatric disorders and death records. In Canada, according to the Canada Health Act, the publicly funded universal health care system covers medical and hospital services provided by physicians for insured residents of Canada, and these databases capture information for all publicly-funded services.Ontario Health Insurance Plan (OHIP) database contains billing records for all insured services claimed by physicians and other health professionals.The Canadian Institute for Health Information Discharge Abstracts Database (DAD) contains abstracts of all discharges from acute, chronic and rehabilitation inpatient facilities [The cilities .National Ambulatory Care Reporting System (NACRS) captures all emergency department visits [The t visits .Registered Persons Database (RPDB) captures death records of everyone insured under OHIP.The HIV-positive participants were included in the analysis if they were 16 years or older, had a valid OHIP number to link to administrative databases, had completed their baseline interview between October 1, 2007 and December 31, 2012, and had one measure of their current level of depressive symptoms available.10) and the rest were administered the 20-item Centre for Epidemiologic Depression Scale (CES-D20). Diagnostic accuracy and reliability of the two screening instruments were verified against Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) criteria for a major depression diagnosis from a recent study conducted in a sample of HIV-positive participants [10 [20 [20 and K10 demonstrate good inter-rater agreement (Cohen’s Kappa Statistic = 0.79) when compared with the DSM-IV-TR criteria for a diagnosis of major depression [20 and K10 have been adopted to identify depressive symptoms in general population [Current depressive symptoms  was identified using two short screening instruments. Due to resource-constraints in several HIV clinics, 61% of HIV-positive participants were administered the 10-item Kessler Psychological Distress Scale  or 10th revision (ICD-10) diagnostic code  having their first episode of depressive symptoms during the five-year follow-up period. A first recurrent case was defined as a participant who was identified with current depressive symptoms at baseline, re-identified with current depressive symptoms beyond two years after baseline, and for whom there was at least one eight-week depression-free periods between baseline and the date of recurrent depressive symptoms  according to the sixteen ICD-9 disease sub-categories . This diWe selected individual explanatory variables and contextual explanatory variables  known to be associated with depressive symptoms. More details about these explanatory variables are provided in All statistical tests were two-sided with statistical significance defined as a p-value<0.05. Analyses were performed using STATA MP v. 13.1 . Our anaWe described overall and explanatory variable-specific prevalence (point and period), recurrence, and incidence for current depressive symptoms. Point prevalence was calculated as the number of cases with current depressive symptoms at baseline. The denominator in point prevalence calculations included HIV-positive participants who had completed their baseline interview between October 1, 2007 and December 31, 2012, and who had one measure for identifying current depressive symptoms . The perWe also used frequency and proportion to determine how many participants had mental disorder co-morbidities in addition to current depressive symptoms at baseline. We used Chi-square or Fisher exact test for bivariable proportion comparison by the participants’ current status of depressive symptoms at baseline.Several explanatory multivariable models were constructed to examine associations between potential explanatory variables and the point prevalence, recurrence and incidence of current depressive symptoms. Explanatory variables entered into and kept in the explanatory multivariate models were based on evidence from prior studies ,58–61. W10 was administered in several HIV clinics with resources constraints, it is possible that the characteristics of participants differed between clinics. In each of our final explanatory models, we controlled for differences between the CES-D20 and K10 as well as the residual difference of participants’ characteristics by the clinic type.As we discussed in the methods section, KWe first constructed modified Poisson regression models with robust error variance to examine factors associated with point prevalence .Second, we constructed Cox proportional hazard regression models to examine factors associated with first recurrent depressive symptoms during the five-year follow-up period among HIV-positive participants with current depressive symptoms. In the final model, we also controlled for the use of antidepressants as identified in the participants’ medical records from clinical abstractions. The definition of antidepressants was based on the first line of antidepressants for managing depression in adults recommended by the Canadian Network for Mood and Anxiety Treatments (CANMAT) Clinical guidelines .Third, we constructed Cox proportional hazard regression models to examine factors associated with the incidence of depressive symptoms among HIV-positive participants who had no history of depression at or prior to baseline.For each Cox model, we tested proportionality assumptions by Schoenfeld residuals against time for each explanatory variable and interactions with linear time and natural log of time. When the variable did not meet the proportionality assumptions, our final multivariable Cox proportional hazard model was stratified by this variable that did not meet the assumptions.Adjusted relative risk (aRR) and 95% confidence intervals (CIs) were reported for the association between each explanatory variable and point prevalence outcome. Adjusted hazard ratio (aHR) were reported for the association between each explanatory variable and incidence and recurrence outcomes.10 (as opposed to the CES-D20) were more likely to be female , more likely to have difficulty in affording housing-related expenses , more likely to have a sense of belonging to their neighbourhoods , and more likely to have control over their housing situation . We therefore included a variable to control for differences between instrument types in our final models.We included 3,816 HIV-positive participants at baseline in our final analysis. The median age of participants was 46 years (Interquartile range: 39–52), and 17% were female. We included 3,816 HIV-positive participants in our prevalence estimation of current depressive symptoms . When weOur point prevalence was 28% (95% CI: 27–29%) at baseline. By the end of the follow-up period, our period prevalence rate was 4.9 per 100 person-years (95% CI: 4.8–5.1). Consistent results were observed between explanatory variables and both prevalence outcomes . For exaWe included 957 HIV-positive participants with current depressive symptoms identified at baseline and had at least two-year follow-up data in our recurrence estimation . When we10.By the end of the follow-up period, 43% of participants with current depressive symptoms at baseline had a recurrent episode. The first recurrence rate of current depressive symptoms was 11.9 per 100 person-years (95% CI: 10.8–13.2). The rate peaked with participants who were 50 or older . A higher recurrence rate was found among participants who were European decent or Indigenous, or who were employed or disabled, students or retired, and among those who lived in poorer housing, had supressed recent viral loads, and/or had completed the KWe included 1,745 HIV-positive participants who were depressive symptoms-free at or prior to baseline and who had at least one year of follow-up data for our incidence estimation . When we10.Among the 1,745 depressive symptoms-free participants, the cumulative incidence of current depressive symptoms was 14% by the end of the follow-up period . The incHIV-positive participants with current depressive symptoms had more additional mental disorders compared to their counterparts without these symptoms . Profiles of other mental disorders by participants’ baseline depressive symptoms status are available in The results of our multivariable analyses for associations between factors and point prevalence, first recurrence and incidence of current depressive symptoms are presented in Tables 10 instrument  are comparable to recent findings (23–26%) from large-scale studies conducted in the U.S. ,72,73. HOur cumulative incidence of depressive symptoms of 14% during follow-up among a depressive symptoms-free cohort at baseline was lower than cumulative incidence of 21% reported in the Malee study . Our incConsistent with prior studies ,58–61, wOur study is the first to provide further examination on factors associated with recurrent depressive symptoms in people living with HIV. Although Malee et al. also explored factors associated to recurrent depressive symptoms, this study was limited to specific factors related to HIV-positive mothers  . AdditioWith regard to factors associated with incidence rate of depressive symptoms, consistent with prior evidence, we found HIV-positive participants who were younger or recreational drug users were more likely to develop incident depressive symptoms –38. UnliOur study is the first to report associations between prevalent, recurrent and incident depressive symptoms and an extensive number of housing and neighbourhood-related contextual factors in people living with HIV. Cross-sectional evidence has shown that poorer and unstable housing conditions are strong explanatory variables of poor mental health outcomes among these people living with HIV ,78. Our Our study has several strengths. First, the OCS is the largest HIV cohort in Ontario, representing about one-fifth of the HIV-positive population in the province; participant characteristics generally represent typical HIV-positive individuals in care . Second,20 and K10), to identify current depressive symptoms, but excellent agreement between these instruments and DSM-IV-TR criteria (for major depression diagnosis) have been demonstrated in this cohort  [Our study has some limitations. First, we relied on screening instruments  . The two81–0.87) .Despite these limitations, the linked data between the OCS and the administrative databases offer useful new information in understanding the epidemiology of depressive symptoms in HIV-positive participants from a longitudinal perspective—current depressive symptoms is highly prevalent and is likely to recur over time, particularly in some high-risk subgroups. Our results support the direction of Ontario’s HIV/AIDS Strategy to 2026, which addresses medical concerns associated with HIV (such as depression) and the social drivers of health in order to enhance the overall well-being of people living with or at risk of HIV. Our findings reinforce the importance of providing effective mental health care and demonstrate the need for long-term support and routine management of depression, particularly for individuals at high risk.S1 Appendix(DOCX)Click here for additional data file.


It is estimated that a total of 120,000 new cancer cases in men and in women in more developed countries could be avoided if exposure to HPV was prevented. We used the nationwide pool of German population-based cancer registry data to estimate the burden of HPV-attributable cancer in this population for the year 2013.Incident cases of cervical cancer, squamous cell carcinoma of the anus, oropharynx (OP), as well as of the vulva, vagina and penis were classified as potentially HPV-associated and identified from the nationwide cancer registry data-pool. We calculated the incidence and proportions of cancer with potentially HPV-associated morphologies. Estimation of the HPV-attributable incidence was based on prevalence-estimates of viral DNA in tumor cells in the respective sites, as provided from the international literature.From the overall 15,936 incident cases of anogenital and OP cancers in 2013, 6239 female and 1358 male cancer cases were estimated to be attributable to HPV. The majority of HPV-attributable cases were contributed by cervical cancer  and oropharyngeal cancer .Even if most HPV-attributable cases were contributed by cervical cancer, anogenital cancer at sites other than the cervix, and oropharyngeal cancer substantially contribute to the burden of HPV-associated cancer. Our nationwide cancer registry data-analyses provide the baseline for long-term population-based monitoring of vaccination-effects on cancer incidence in Germany. Persistent infection with Human Papillomavirus (HPV) is considered a necessary cause in the etiology of virtually all cases of invasive cervical cancer . In GermMonitoring of HPV-associated disease remains crucial as two prophylactic HPV-vaccines have been licensed in Germany in 2006 and are recommended since 2007 by the German Standing Committee on Vaccination (STIKO). In 2014, the recommended age for vaccinating girls was lowered from 12–17 years to 9–14 years . High vaWe used the nationwide pool of German population-based cancer registry data to assess the baseline incidence of HPV-associated cancer in our population for the year 2013, yet before quantifiable effects from HPV-vaccination on cancer incidence can be anticipated. Thus, our analyses provide the basis for a continuous monitoring of the effects of HPV-vaccination on cancer incidence at the population-level. This also applies to HPV-associated cancer in men, as a positive effect from vaccinating girls on HPV-related outcomes in men is expected . PotentiCases of cervical cancer with epithelial, squamous cell, basal and transitional cell origin, adenocarcinomas and cystic carcinomas as well as mixed tumors; squamous carcinoma of the anus and oropharynx, the vulva, vagina and penis were classified as potentially HPV-associated carcinoma Table . The IntINK4a overexpression) - a validated and widely used HPV-detection algorithm [INK4a overexpressive keratinizing or warty-basaloid vulvar SCC [Pathophysiological knowledge and epidemiologic evidence strongly suggest that the presence of HPV-infection in tumor material is sufficient to infer that HPV-infection caused the cancer. Estimation of the population attributable fraction (PAF) due to HPV was therefore based on the prevalence of viral DNA in squamous cell carcinoma (SCC) cells in the respective sites, as provided from the international literature, preferably from most recent metaanalyses and large tissue sample studies , 11–15. lgorithm . The oroNamely, all cases of cervical cancer and 88–90% of anal carcinoma with HPV-associated morphologies were considered HPV-attributable. An HPV-attributable proportion of 32% was assumed in penile cancer with HPV-associated morphologies, whereas 81% of vaginal, 18% of vulvar, and 16% of OP cancer with potentially HPV-associated morphologies were considered HPV-attributable . Continuous registration has commenced in 1970 in the Saarland, but nationwide coverage has not been reached before 2009, when the population-based cancer registry of Baden-Württemberg was implemented. According to our current nationwide estimation, the degree of cancer registration across Germany is high, and 96%  of the estimated 482,473 new cancer cases in 2013 have actually been recorded in the registries (data not shown). For our analyses, we retrieved incident cases of cervical cancer, penile cancer, vulvar cancer, vaginal cancer, anal cancer, and oropharyngeal cancer from the nationwide data pool for 2013. Site, behavior and histology are coded according to the International Classification of Diseases for Oncology (ICD-O-3).For the purpose of monitoring, we provide the number of newly diagnosed cases, proportions and rates of cancer with potentially HPV-associated morphologies. Morphology codes of potential HPV-association are shown in Table Incidence rates from 2013 were age-standardized to the old European standard population (ASIR). 95% confidence intervals of ASIR were calculated using the binomial approximation according to the recommendation of IARC . CalculaIn 2013, 5564 male incident cases and 10,372 female incident cases of invasive anogenital and oropharyngeal cancers were registered in Germany. Overall 9573 incident female cancer cases with potential HPV-associated site and morphology were extracted from the pooled data. Amongst them were 6239 incident cases considered to be attributable to HPV; 4422 cervical cancers , 927 anal cancers, 467 vulvar cancers, 223 vaginal cancers, and 200 oropharyngeal cancers. Among the 5246 incident male cancer cases with potential HPV-associated site and morphology, 1358 cases were attributed to HPV. Oropharyngeal cancer constituted the largest group  followed by anal cancer (496 cases) and penile cancer (225 cases). Table Overall, a proportion of 1.6% of an estimated 482,473 new cancer cases in Germany in 2013 was considered HPV-attributable. The number of 9573 female cancer cases and 5246 male cancer cases with potential HPV-associated site and morphology assessed corresponds to approximately 3.1%  of the overall cancer burden in Germany in the same year. The proportion of incident cases with potentially HPV-associated morphology that were redistributed from cases morphologically not otherwise specified ranged between 3.9%  and 9.6% (vagina).The most common cancers in the female and male population were cervical cancer in women and oropharyngeal cancer in men. Non-cervical anogenital cancer with HPV-associated morphology were rare in the German population. Anal cancer with an HPV-associated morphology was however more common in women than in men Table .Table 3AThe age-distribution largely differed by site Fig.  and b. TCorrespondingly, crude incidence rates (IR) of cervical cancer were highest in the age groups of 35–49 years and 50–64 years  and hereafter declined  and time, the actual HPV-prevalence among cancer cases in Germany might deviate from the prevalence estimates we presumed. In oropharyngeal cancer, previous studies from Germany that were included in a recent metaanalysis  and one The quality of epidemiological cancer registry data limits registry-based studies in general, and also needs to be considered in our study: An aspect of uncertainty is introduced by potentially incomplete registration of incident cases in some registries, leading to underestimation. However, nine German federal states had achieved sufficient data quality and completeness to be included in the IARC publication “Cancer in five continents (CI5)” for the years 2003–2007, which can be regarded as a reference for high quality registries . Since 2In recent studies of penile, anal, vaginal and vulvar cancer, the histological term “warty” or “basaloid/warty” carcinoma has been used to characterize HPV-associated carcinoma –14. OnlyDue to missing information on site specific HPV-prevalence in the general German population  our PAF-It should be mentioned, that our estimates only take invasive tumors into account, while especially for the cervix, a large number of high-grade dysplastic lesions contribute to the HPV-related disease burden, demanding invasive treatment. Similar to other published estimates on HPV-related disease burden, we did not consider these cases in our analyses.HPV-associated anogenital cancer and oropharyngeal cancer substantially contribute to the burden of cancer. In the United States of America (US), cancer with HPV-associated site and morphology account for 2% of male cancer cases and 3.3% of female cancer cases . Our estFor long-term monitoring of time trends (over 2–4 decades), a focus on cancer sites with high HPV-attributable fractions in younger age-groups (35–49 years) might be reasonable, though effects can first be expected to become observable in the more distant future: In Germany, HPV vaccination commenced in 2007 for 12–17 year old girls. As the oldest vaccinated women from the birth cohort of 1990 will not reach age 35 until 2023, it will be 2038 before the 35–49 age-group consists entirely of potentially vaccinated women.The attributable fraction method may however not be suitable for monitoring time trends in the incidence of HPV-attributable cancers for sites with low attributional fractions, such as vulvar cancer and the oropharyngeal cancers group. Using vulvar cancer as an example, 18% of all vulvar cancers diagnosed in 2013 were attributed to HPV infection; 82% were attributed to other causes. If HPV vaccination decreases the incidence of HPV-attributable vulvar cancer in future years, this will hardly become apparent in the overall estimates, and the effect of the vaccine especially in low-PAF cancers in absolute terms will be underestimated without regular monitoring of the prevalence of HPV DNA in cancer cells. A decreasing proportion of cancer with HPV-associated morphology at respective sites would be expected. The newly emerging clinical cancer registries in Germany  might pOur estimates of the cancer burden at HPV-related sites along with the HPV-attributable fractions provide a baseline assessment of the HPV-related cancer burden in Germany and contribute to future evaluation of long-term HPV-vaccination effects on the population level. With population-based clinical cancer-registration, future monitoring of HPV-associated cancer in Germany is feasible with more accuracy and precision than using global estimates.


We report here the first genome sequence of infectious laryngotracheitis virus isolated in Peru from tracheal tissues of layer chickens. The genome showed 99.98% identity to the J2 strain genome sequence. Single nucleotide polymorphisms were detected in five gene-coding sequences related to vaccine development, virus attachment, and viral immune evasion. Infectious laryngotracheitis is an avian respiratory disease which is caused by infectious laryngotracheitis virus (ILTV), and clinical signs vary from coughing, conjunctivitis, and gasping to expectoration of bloody mucus and death .Whereas genomic database analysis allowed the identification of an ILTV field strain in Australia as a causative agent of outbreaks as a result of recombination between attenuated vaccine viruses , genomicILTV was reported for the first time in Peru by the Servicio Nacional de Sanidad Animal (SENASA) in 2008 . ILTV inIn December 2014, an ILT outbreak occurred at a farm in Chincha Alta, Ica, Peru. An ILTV field strain named VFAR-043 was isolated from tracheal tissues and propagated onto the chorioallantoic membrane (CAM) of specific-pathogen-free eggs . This ande novo using NextGene , using a HiSeq 2000 platform. The reads were assembled NextGene , with a NextGene . BLAST  in 5 gene-coding sequences (CDS) of VFAR-043 in comparison to the SA2 strain. We detected 4 nsSNPs in glycoprotein B (gB) (UL27), a protein which is associated with viral attachment to cells ; 2 nsSNPTo our knowledge, this work presents the first ILTV genome sequence reported in South America; therefore, this study will allow us to clarify ILTV epidemiology in Peru and to contribute to the knowledge of this virus in this region.MG775218. The version described in this paper is the first version, MG775218.1.This full-genome sequence of the VFAR-043 strain has been deposited to GenBank under accession no.


ARQ 197-215 was a randomized placebo-controlled phase II study testing the MET inhibitor tivantinib in second-line hepatocellular carcinoma (HCC) patients. It identified tumor MET as a key biomarker in HCC.MNNG-HOS transforming gene) and circulating  biomarkers in second-line HCC. Tumor MET-High status was centrally assessed by immunohistochemistry. Circulating biomarkers were centrally analyzed on serum samples collected at baseline and every 4-8 weeks, using medians as cut-off to determine High/Low status. Tumor MET, tested in 77 patients, was more frequently High after (82%) versus before (40%) sorafenib. A significant interaction (p = 0.04) between tivantinib and baseline tumor MET in terms of survival was observed. Baseline circulating MET and HGF (102 patients) High status correlated with shorter survival , while the association between AFP (104 patients) or VEGF (103 patients) status and survival was non-significant.Aim of this research was to study the prognostic and predictive value of tumor (MET, the receptor tyrosine kinase encoded by the homonymous Conclusions: Tumor MET levels were higher in patients treated with sorafenib. Circulating biomarkers such as MET and HGF may be prognostic in second-line HCC. These results need to be confirmed in larger randomized clinical trials. Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related death , and sorMNNG-HOS transforming gene (MET) encoding the receptor tyrosine kinase for hepatocyte growth factor (HGF) is involved in cancer progression and metastasis formation, and suggests poor prognosis in early stage and second-line HCC patients [The patients -8. Hypoxpatients , 9, 10, patients , 12. METpatients . MET is patients , 15.Tivantinib is an oral, ATP-independent inhibitor of MET, binding to its inactive form , 17; itsFollowing promising phase I study results with tivantinib as monotherapy and in combination with sorafenib in HCC , 33, theConsidering the lack of accepted biomarkers in HCC and realizing the necessity of placebo-controlled studies, we analyzed the prognostic and predictive values of tumor (MET) and circulating , vascular endothelial growth factor (VEGF)) biomarkers in the ARQ 197-215 study.Tumor samples of sufficient quality for tumor MET analysis at immunohistochemistry (IHC) were submitted for a total of 77 patients, 49 who were randomized to tivantinib and 28 to placebo [N = 55), and 82% if obtained after sorafenib  were found to be MET-High. Biopsy date was known for 72 samples. When samples were analyzed with respect to prior therapy, the chance of being MET-High was 40% if the biopsy was obtained before sorafenib . Tivantinib was ineffective in patients with MET-Low tumors , suggesting that tivantinib may offset the negative prognostic impact of high MET expression . Correlation between MET and other baseline characteristics was not detected except for a slight overlap between MET-High patients and patients with a baseline AFP higher than the median.The test for interaction between treatment and MET status showed a statistical significance at an alpha level of 0.05 for OS . The median MET concentration at baseline was 13.26 ng/mL, with a range of 1.29-49.8 ng/mL. Eighty-six patients  who were evaluable for survival were also evaluable for changes in circulating MET over the course of the study. Baseline characteristics were generally balanced between groups, except for higher prevalence of hepatitis C virus (HCV) infection  in the circulating MET-High compared to the MET-Low group  and 3.8 months on placebo (N = 15), . The OS in circulating MET-Low patients was 7.5 months on tivantinib (N = 32) and 9.4 months on placebo (N = 19), . Such an advantage was evident by week 8 of therapy . No such trend was observed in patients receiving placebo . The baseline median HGF concentration was 2307 pg/mL, with range of 421-58080 pg/mL. Eighty-six patients  who were evaluable for survival were also evaluable for changes in circulating HGF over the course of the study. Baseline characteristics were generally balanced between groups  but not for patients on placebo . Furthermore, the test for interaction shows no correlation between HGF and OS benefit with tivantinib.Patients with a baseline HGF lower than the median survived longer than patients with a higher baseline HGF regardless of the therapy . No difference in OS was observed when comparing patients with and without HGF reduction on tivantinib  or placebo . In patients with stable disease past the first tumor assessment, median change from baseline HGF was +28.2%  regardless of treatment.Patients with a reduction over time by at least 10% in circulating HGF survived longer than patients with no or minimal reduction . The baseline median AFP concentration was 186 IU/mL, with range of 1.5-440008 IU/mL. Baseline characteristics were generally balanced between groups . To understand if higher AFP values have a greater prognostic impact, an additional analysis was performed using an arbitrary cut-off value equal to the AFP 75th percentile (third quartile (Q3); 3507.50 IU/mL) resulting in longer OS for patients with baseline values lower than Q3 .A trend towards better outcome was seen in patients with a baseline AFP lower than the median, regardless of treatment allocation  had baseline values ≥20 IU/mL, had samples at multiple time points, and were evaluable for OS; therefore, no analysis was performed on AFP changes over time.versus placebo by any AFP status was comparable, with the test for interaction resulting non-significant.Survival of patients on tivantinib N = 51), 5.0 months in patients with VEGF-High (N = 52) ; no trend was observed by treatment arm. Survival of patients with a VEGF reduction over time by at least 10% tended to be longer than survival of patients with no or minimal reduction , regardless of treatment.Circulating VEGF was measured in 103 patients also evaluable for OS . Median baseline circulating VEGF was 160 pg/mL. Baseline characteristics were generally balanced between groups  enrolled only patients with tumor MET-High HCC. Such studies may clarify the role of MET, HGF, and AFP in this patient population.MET expression was evaluated by IHC assay, on the most recent sample available, using the CONFIRM™ anti-total MET(SP44) antibody , analyzed at an independent Clinical Laboratory Improvement Amendments (CLIA) certified laboratory by a board certified pathologist after randomization and prior to study un-blinding. MET-High was defined as a majority (≥50%) of tumor cells with moderate or strong (2+ or 3+) staining intensity per ArQule's protocol. A negative control slide from each patient sample was run as well as positive control from colon adenocarcinoma ensuring that proper antibody and reagent dispensing took place. Additionally, results of MET testing were reported as an H-score, calculated by multiplying the percentage of cells staining by the intensity of the stain .Serum biomarker concentrations were measured by ICON Central Labs, Farmingdale, NY, using commercial materials.MET, HGF and VEGF serum samples were collected before the first dose on cycle 1 day 1, and post dose every 4 weeks thereafter, and at treatment end. One blood collection tube was used for MET/HGF testing and another for VEGF testing.For circulating MET measurement, Kamiya Biomedical K-ASSAY Human MET ELISA kit , Automated ELISA System (Dynex Technologies), and the Spectramax reader (Molecular Devices) were used. The Quantikine Human HGF Immunoassay  was used to measure HGF levels in serum using the Spectramax reader (Molecular Devices). For VEGF, QuantiGlo Human VEGF immunoassay kit from R&D  using the Spectramax Plus 384 reader was used.AFP serum samples were collected at screening/pre-study, and every 8 weeks after randomization, as well as at the end of treatment. AFP levels were tested using automated analyzer Immulite 2000 .th percentile. As for the changes in biomarker concentrations, the best change (highest reduction or the lowest increase) during the treatment period for each patient was calculated as the percent change from baseline, and 10% was arbitrarily chosen as cut-off value. Survival analysis by biomarker reduction was also performed restricting the sample to patients who survived to a pre-specified arbitrary landmark time of 8 weeks after the date treatment was started. This latter analysis was specifically undertaken to eliminate guarantee-time bias and was proposed by others in similar settings [All statistical analyses were performed using SAS statistical software. Biomarker baseline values and their changes over the course of treatment were dichotomized and analyzed with respect to OS probability. Median OS and the 95% CI were estimated using the Kaplan-Meier method and the logrank test was used to compare the survival distributions. The Cox regression model was used to obtain the HR. The interaction between biomarkers and the treatment was evaluated using the Cox proportional hazards model with an interaction term. Multivariate analysis was not attempted due to the small sample size of available biopsies for tumor MET biomarker assessment. The correlation between the biomarkers was assessed using Fisher's exact test. The median concentrations of the serum baseline biomarkers were used as the cut-off values to differentiate the respective Low and High groups for the study of prognostic and predictive effects. However, in the case of AFP, an additional arbitrary cut-off was set at the 75settings , 48.