Worldwide, depression is rated as the fourth leading cause of disease burden and is projected to be the second leading cause of disability by 2020. Annual depression-related costs in the United States are estimated at US $210.5 billion, with employers bearing over 50% of these costs in productivity loss, absenteeism, and disability. Because most adults with depression never receive treatment, there is a need to develop effective interventions that can be more widely disseminated through new channels, such as employee assistance programs (EAPs), and directly to individuals who will not seek face-to-face care.This study evaluated a self-guided intervention, using the MoodHacker mobile Web app to activate the use of cognitive behavioral therapy (CBT) skills in working adults with mild-to-moderate depression. It was hypothesized that MoodHacker users would experience reduced depression symptoms and negative cognitions, and increased behavioral activation, knowledge of depression, and functioning in the workplace.A parallel two-group randomized controlled trial was conducted with 300 employed adults exhibiting mild-to-moderate depression. Participants were recruited from August 2012 through April 2013 in partnership with an EAP and with outreach through a variety of additional non-EAP organizations. Participants were blocked on race/ethnicity and then randomly assigned within each block to receive, without clinical support, either the MoodHacker intervention (n=150) or alternative care consisting of links to vetted websites on depression (n=150). Participants in both groups completed online self-assessment surveys at baseline, 6 weeks after baseline, and 10 weeks after baseline. Surveys assessed (1) depression symptoms, (2) behavioral activation, (3) negative thoughts, (4) worksite outcomes, (5) depression knowledge, and (6) user satisfaction and usability. After randomization, all interactions with subjects were automated with the exception of safety-related follow-up calls to subjects reporting current suicidal ideation and/or severe depression symptoms.At 6-week follow-up, significant effects were found on depression, behavioral activation, negative thoughts, knowledge, work productivity, work absence, and workplace distress. MoodHacker yielded significant effects on depression symptoms, work productivity, work absence, and workplace distress for those who reported access to an EAP, but no significant effects on these outcome measures for those without EAP access. Participants in the treatment arm used the MoodHacker app an average of 16.0 times (SD 13.3), totaling an average of 1.3 hours (SD 1.3) of use between pretest and 6-week follow-up. Significant effects on work absence in those with EAP access persisted at 10-week follow-up.2 = .021) among employed adults at 6-week follow-up when compared to subjects with access to relevant depression Internet sites. The app had stronger effects for individuals with access to an EAP . For all users, the MoodHacker program also yielded greater improvement on work absence, as well as the mediating factors of behavioral activation, negative thoughts, and knowledge of depression self-care. Significant effects were maintained at 10-week follow-up for work absence. General attenuation of effects at 10-week follow-up underscores the importance of extending program contacts to maintain user engagement. This study suggests that light-touch, CBT-based mobile interventions like MoodHacker may be appropriate for implementation within EAPs and similar environments. In addition, it seems likely that supporting MoodHacker users with guidance from counselors may improve effectiveness for those who seek in-person support.This randomized effectiveness trial found that the MoodHacker app produced significant effects on depression symptoms  Major depressive disorder is one of the most prevalent mental conditions to afflict adults in the United States, with estimates in the United States for major depression of 16.6% for lifetime occurrence and 6.7% for a 1-year period . WorldwiBased on data from 2010, depression-related costs in the United States exceeded US $210.5 billion, with employers incurring US $102 billion in losses due to presenteeism (US $78.7 billion), absenteeism (US $23.3 billion), and disability, and another US $98.9 billion incurred as direct medical costs . Each yeBecause most adults who suffer from depression never receive treatment , there iThe Coping with Depression (CWD) cognitive behavioral therapy (CBT) skills-training program ,22 is bad=0.28 to 0.62, depending on the outcome measure used [The CWD approach has been validated for use with a variety of age, gender, and race/ethnic groups and using a variety of delivery methods, including as a guided self-help intervention . Based oCognitive and behavioral approaches to mood have evolved from a focus on treating disorders to including an emphasis on promoting positive emotion and experiences. Positive psychology interventions (PPIs), such as mindful self-awareness, gratitude expression, and identifying and utilizing strengths, have been shown to impact both well-being and depressive symptoms -31. Mindd=0.44-1.90 for clinician-supported, and d=0.21-0.70 for self-guided, interventions [d=0.42-0.65) than for populations with mild-to-moderate symptoms (d=0.30-0.53) [Internet-delivered CBT-based programs for depression symptom management have been shown to be effective both for adults with major depressive disorder and those with elevated, but subthreshold, depression symptoms, although one recent review identified concerns with dropout and lack of long-term effects -45. Tradventions -45,47,4830-0.53) .To date, very few randomized clinical trials of mobile apps targeting depression have been published in the peer-reviewed literature . We founThe SuperBetter mobile Web app was evaluated in a three-arm randomized controlled trial, with results showing the original, “general” version of SuperBetter—with activities focused on self-esteem and social support—more effective at reducing depressive symptoms over both a combined SuperBetter-plus-CBT and positive psychology strategies condition and the waiting list condition. Ly et al  found noThe CWD-based Overcoming Depression on the Internet program provided early evidence that a Web-based program with reminders was effective in reducing depression symptoms, particularly among individuals with mild-to-moderate depression ,58. SpekInternet delivery of mental health interventions offers individuals with online technology broad access to evidence-based treatment. Mobile apps offer an additional channel to increase access further, both as self-guided tools and as those supported by counseling professionals, such as EAPs. Globally, use of mobile and broadband mobile services is rising, while fixed broadband service is slowing, especially in developing countries . WorldwiMobile interventions offer the potential for anytime, anywhere convenience and the ability to promote regular use of behavioral and cognitive self-management strategies known to impact mood. Consumers express high interest in, and willingness to use, mobile phones and short message service (SMS) text messaging to monitor and manage symptoms and cite essential considerations such as ease of use, privacy, and security -66. ProvThe MoodHacker mobile intervention is one of few clinically validated CBT-based depression self-management mobile apps currently available. MoodHacker is designed to directly activate key cognitive and behavioral skills from the validated CWD program  and posiTo demonstrate the efficacy of this light-touch, mobile, Web CBT-based experience, we compared MoodHacker as a fully self-guided intervention to an alternative-treatment control group that received an email with links to vetted online information about depression. It was hypothesized that MoodHacker users would experience reduced depression symptoms and negative cognitions, and increased behavioral activation, knowledge of depression, and functioning in the workplace. This study of the MoodHacker intervention extends the evidence base for CBT-based mobile interventions for adults with mild-to-moderate depressive symptoms. Because the MoodHacker mobile Web app was designed primarily to target employees who present with depression through their EAP, the extent to which the program effects generalize to users with and without access to EAP support was an important research question.The efficacy of the MoodHacker mobile Web app intervention was assessed with a randomized controlled trial  with two factors: condition and EAP access . See Inclusion criteria for participation were defined as follows: (1) 18 years or older, (2) mild-to-moderate depressive symptoms as measured by the Patient Health Questionnaire-9 (PHQ-9) (score of 10-19), (3) not currently suicidal or meeting criteria for bipolar or schizoaffective disorder, (4) employed at least part time, (5) English speaking, and (6) have access to a high-speed Internet connection. Eligibility was assessed using a two-stage screening protocol. In total, 3064 individuals completed an online screening survey, which included questions on demographics, technology access, and depression symptoms , as well as brief screening for bipolar and schizoaffective disorders. Of those, 856 (27.94%) qualified for secondary telephonic screening to more fully assess their depression symptoms with the PHQ-9 and to confirm their assessments for bipolar and schizoaffective disorders. Of those 856 who qualified, 294 (34.3%) individuals failed to complete the secondary screening, 205 (23.9%) did not meet the PHQ-9 criteria, 44 (5.1%) endorsed suicidal ideation or showed symptoms of bipolar or schizoaffective disorder, and 13 (1.5%) were dropped due to suspicion of fraud (see below). A total of 300 individuals out of 856 (35.0%) from 37 US states completed the online baseline assessment and were randomly assigned to one of two conditions: treatment or alternative care. See Participants were recruited from August 2012 through April 2013 in partnership with several organizations, including Chestnut Global Partners EAP as our primary recruitment partner. Non-EAP recruitment partners included Hope to Cope, Esperanza, Mental Health America, National Alliance on Mental Illness, LIVESTRONG, eHow, other eHealth websites, Chamber of Commerce offices, employee support organizations, and Craigslist. Outreach was conducted via the Chestnut EAP call center, print ads, online postings and ads, email listservs, and flyers. All interested potential participants were directed to an informational website that described the broad characteristics of the study’s purpose, activities, and compensation, concluding with an online screening survey.All subjects participated fully online from the location(s) of their choice, using their own Internet-capable computers and mobile devices. All self-report, online screening and assessment data were collected via encrypted websites. Upon prequalification based on the initial screening survey, research staff conducted telephone interviews with potential participants to determine eligibility per the inclusion criteria referenced above. Potential participants who reported current suicidal ideation and/or bipolar or schizoaffective disorder during screening were offered appropriate resources according to an IRB-approved crisis protocol and were excluded from the study. The number of participants who self-identified with bipolar disorder symptoms in the initial screening survey was elevated due to suspected fraudulent individuals attempting to qualify with severe symptoms.Throughout the study, individuals who reported current suicidal ideation and/or severe depression symptoms (PHQ-9 > 19) were contacted by telephone and offered appropriate resources according to an IRB-approved crisis protocol. Calls were made to 114 individuals  and all remained in the study. No subjects reported suicide risk severe enough to transfer to a suicide hotline. Nor did any subjects report any adverse events related to the use of the MoodHacker app via email or during follow-up calls. Although research assistants were aware of group assignment, all other interactions with subjects were delivered by emails that were standardized across groups and fully automated to avoid differential interactions by group assignment. All other research team members were blinded and, aside from crisis calls, no research team members had direct interaction with subjects after randomization.Lack of direct contact with study participants in a fully online study lends itself to potential participants self-reporting false information to qualify . To identify these individuals, demographic and contact data were cross-checked for fraudulent information against other individuals in the study database, as well as in our database of over 20,000 records of previous Internet study applicants. Those suspected of submitting fraudulent data were dropped from the study prior to randomization.To enhance sample representativeness in each experimental condition, qualified participants were blocked on race/ethnicity and then randomly assigned within each race/ethnicity block to condition—treatment or alternative care—using the random number function in our subject database. Emails indicating group assignment and linking participants to the online informed consent form were auto-generated in the database and sent to participants by a research assistant. Upon completion of the consent form, they were immediately linked to the online baseline self-assessment. Participants also completed online follow-up self-assessments at 6 weeks and 10 weeks after baseline. Participants were compensated US $50 per completed assessment.After completing the baseline assessment, participants in the treatment condition were emailed a link to the MoodHacker mobile Web app and instructed to use the app for the next 6 weeks. Participants in the alternative care condition were sent an email with links to credible online resources about depression and instructed to review it on their own schedule for the next 6 weeks. Alternative care participants were offered use of the MoodHacker app upon completion of the 10-week assessment.The MoodHacker responsive mobile Web app was designed to educate users about depression and the benefits of CBT-based strategies to improve mood self-management and to activate (1) daily mood and activity monitoring, (2) increased engagement in positive behavioral activities, (3) decreased negative thinking and increased positive thinking, (4) increased practice of gratitude, mindfulness, and strength-based cognitions and behaviors, and (5) daily practice of these skills to improve depression symptoms and increase resilience to future mood disturbances.Articles & Videos library. Daily emails  depression symptoms, (2) behavioral activation, (3) negative cognitions, (4) worksite outcomes, (5) knowledge, and (6) user satisfaction and program usability. The primary outcome measure was depression symptoms, which was the target of the intervention. Secondary or exploratory measures included the following: (1) potential mediators  and (2) potential worksite outcomes that may be influenced by improvement in worker depression. Participants completed self-report assessments at each of the assessment points: baseline, 6 weeks, and 10 weeks.Demographic data were collected during the screening process, including the following: (1) gender, (2) age, (3) race/ethnicity, (4) marital status, (5) highest completed education, (6) household income, and (7) employment status.Depressive symptomatology was assessed at each assessment point using the self-reported PHQ-9   to assesHow actively individuals are taking care of themselves, including making positive life choices, was expected to increase as a result of the intervention. Behavioral activation was measured using the Behavioral Activation for Depression Scale (BADS) Short Form ,78. ThisChange in negative thinking was assessed using the Automatic Thoughts Questionnaire-Revised (ATQ-R) scale Short Form . A 12-itParticipants were assessed for increase in knowledge about depression. The scale consisted of 14 multiple-choice items developed for this study based on the 12 learning objectives addressed in the MoodHacker articles and videos. Higher scores indicate higher knowledge. Test/retest reliability over a 6-week interval for this scale was .65.Worker productivity was assessed using the Work Limitations Questionnaire (WLQ)  -82. The Productivity loss due to work absence was assessed using the two-item WLQ Work Absence Module, which asks about the number of full days and part days missed in the last 2 weeks due to health problems or medical care . The perWorksite outcomes were also assessed using the Workplace Outcome Suite (WOS)  . This inAt 6 weeks, treatment participants completed the System Usability Scale, which is a quantitative measure of program ease of use . The scad=0.34 or larger . A recent meta-analysis of Internet-based CBT interventions for depression [d=0.32 for change in depressive symptoms and the mean effect size obtained in a randomized pilot study evaluating an abbreviated prototype of this app was d=1.05. Thus, this sample size provided adequate statistical power to detect the anticipated effects for the primary outcomes of interest.Statistical power calculations for the analysis of covariance (ANCOVA) indicated that a sample size of 300 yielded sufficient power (>.80) to detect a condition effect of Cohen’s pression  had founP<.05, two-tailed, for all tests.Univariate effects of intervention condition, EAP access, and their interaction on outcome measures were examined using between-subjects ANCOVA, adjusting for pretest outcomes. These analyses were conducted to evaluate effects on outcome measures assessed at both 6-week and 10-week follow-up. If the condition by EAP access interaction was significant for an outcome measure, separate subpopulation ANCOVA analyses were conducted on that outcome measure for subjects with and without EAP access. We explored dose-response relationships and self-monitoring participation within the treatment group by correlating process indicants with change in outcome measures. All subjects were included in intent-to-treat (ITT) analyses at each follow-up. Prior to conducting these analyses, we employed the single imputation procedure available in SPSS, version 21.0 (IBM Corp) to account for missing data. Alpha was set to t tests were conducted on categorical and continuous measures, respectively. The groups did not significantly differ on any demographic characteristics or pretest outcome measures. See The expectation of baseline equivalency due to random assignment of groups was examined. The treatment and alternative care groups were compared on demographic characteristics and outcome measures collected at pretest. Contingency table analyses and The extent to which attrition threatened the internal and external validity of the study was evaluated using contingency table analyses and analysis of variance (ANOVA). Participants who completed each of the follow-up assessments were compared to those who did not complete that follow-up with respect to demographic characteristics and pretest outcome measures. We also conducted analyses to test whether outcome variables were differentially affected across conditions by attrition. These latter analyses examined the effects of condition, attrition status, and their interaction on pretest outcomes. Examination of attrition between pretest and 6-week follow-up revealed only 10 out of 150 (6.7%) treatment participants did not complete the assessment compared to 4 out of 150 (2.7%) alternative care participants. Only 10 out of 150 (6.7%) treatment participants did not complete the 10-week follow-up assessment compared to 5 out of 150 (3.3%) alternative care participants. Attrition rates did not significantly differ by condition. Moreover, we found no statistically significant differences in demographic characteristics or baseline outcomes by attrition status, nor did we find any statistically significant interactions between attrition and condition predicting baseline outcomes, suggesting that attrition was not systematic.P=.003), consisted of fewer Hispanics (P=.01), were more likely to have full-time employment (P=.001), had a higher level of education (P=.047), and had a greater income (P=.001).Analyses compared baseline demographic data of subjects with EAP access versus those without EAP access. Subjects with EAP access had significantly more children . However, a statistically significant condition-by-user-EAP-access interaction effect was also obtained , indicating differential program effects depending upon subjects’ access to an EAP. Separate subpopulation analyses indicated significant positive program effects for subjects with EAP access  and no program effects for subjects without EAP access . From pretest to 10-week follow-up, the condition-by-EAP-access interaction effect was not statistically significant , so subpopulation analyses were not indicated. The ANCOVA with the full sample found that there were no program effects  at 10-week follow-up.From pretest to 6-week follow-up, the ANCOVA with the full sample found statistically significant program effects on depression symptoms (PHQ-9) , ATQ-R , and knowledge . The condition-by-EAP-access interaction effects were not statistically significant on any of the mediator measures, indicating that there was no need for subpopulation analyses. From pretest to 10-week follow-up, the condition-by-EAP-access interaction effects were not statistically significant on any of the mediator measures. The ANCOVA with the full sample found statistically significant program effects on BADS , but not on ATQ-R  or knowledge .From pretest to 6-week follow-up, the ANCOVA with the full sample found statistically significant positive effects for the program on each mediator measure: BADS , the WLQ work absence measure , and the workplace distress measure . Consequently, subpopulation analyses were carried out for these measures only. The ANCOVA with the full sample at 6-week follow-up found statistically significant program effects on WLQ work absence . No statistically significant program effects were found on the following: WLQ productivity loss , WOS absenteeism , WOS presenteeism , WOS engagement , or WOS life satisfaction . However, separate subpopulation analyses at 6-week follow-up on the WLQ productivity loss measure indicated significant positive effects of the program for subjects with EAP access  and no program effects for subjects without EAP access . Similarly, subpopulation analyses at 6-week follow-up on the WLQ work absence measure indicated significant positive effects of the program for subjects with EAP access  and no program effects for subjects without . Lastly, the WOS workplace distress measure indicated significant positive effects of the program for subjects with EAP access  and no program effects for subjects without .From pretest to 6-week follow-up, the condition-by-EAP-access interaction effects were statistically significant on the WLQ productivity loss measure . Consequently, subpopulation analyses were carried out for this measure only. The ANCOVA with the full sample at 10-week follow-up found statistically significant program effects only on the WLQ work absence measure . Separate subpopulation analyses at 10-week follow-up on the WLQ work absence measure indicated significant positive effects of the program for subjects with EAP access  and no program effects for subjects without EAP access .From pretest to 10-week follow-up, the condition-by-EAP-access interaction effects were statistically significant on the WLQ work absence measure  for a total duration of 1.3 hours  between pretest and 6-week follow-up. The average rating of program satisfaction was 4.6 (SD 1.0) on a 6-point scale, indicating that the participants were mostly satisfied with the intervention. Participants also completed the System Usability Scale  at the 62 = .021 for the full population and partial eta2 = .093 for those with EAP access, which convert to approximately Cohen’s d=0.30 and 0.64, respectively) are comparable to those reported for previous meta-analyses of self-guided, Internet-based CBT programs (Cohen’s d=0.21-0.31) [This randomized effectiveness trial examined the effect of the MoodHacker mobile Web app on depression symptoms, important cognitive and behavioral mediators, and workplace outcomes among employed adults with and without access to EAP services. The MoodHacker app produced significant effects from pretest to 6-week follow-up for all subjects compared to alternative care subjects on (1) the clinical outcome measure , with much stronger results among those with EAP access, (2) all three mediating outcome measures , and (3) one of the worksite outcome measures . Among subjects with EAP access, larger effects were found on depression symptoms, productivity loss, work absence, and workplace distress. Significant effects were maintained at 10-week follow-up for work absence for all subjects, with much stronger results among those with EAP access. The effect sizes on depression symptoms in this study  ,44.These findings suggest that the approach used to activate CBT-based skills in MoodHacker was effective and begin to build an evidence base for light-touch, CBT-based mobile apps for depression self-management. Because program effects were small-to-medium size, modifications to increase the potency of the intervention are warranted. Evidence from prior online CBT-based programs suggests that supporting the mobile intervention with counselors, such as those working in EAPs, is likely to increase both adherence to the app and efficacy in reducing depression symptoms ,43,47,89At the 10-week assessment, only effects on work absence remained significant. Effects on depression symptoms were no longer significant, possibly because the daily emails ended after 6 weeks; the emails included prompts to view content, introduction of key concepts and skills, tips to optimize use of the program, and encouragement to track mood and activities. Although our results are consistent with a recent meta-analysis of computerized CBT programs showing that results of computer-based CBT programs typically attenuate over time , evidencThe inclusion of subjects without access to EAP services provided an important opportunity to evaluate program efficacy with this population and to compare program efficacy for subjects with and without EAP access. It was expected that the subjects with access to EAP services who chose to participate in this study were likely to be quite similar to real-world individuals who might elect to use a mobile or online program in conjunction with those services. Thus, these subjects provided a “real-world” effectiveness trial. The effect sizes found on depression symptoms, work absence, and productivity loss among the targeted EAP population are quite encouraging and suggest that EAPs offering use of the MoodHacker app may reasonably expect to find significant depression-related improvements in their employee populations. Conclusions regarding program effects for non-EAP individuals are less clear.In many cases, the subjects without EAP access were recruited without the endorsement of a trusted partner, most notably when recruitment was through entities such as eHow, Chambers of Commerce, eHealth websites, and especially Craigslist. This raises questions regarding the motivation of these subjects for participation. For example, participants who presented to their EAP with depression symptoms and were willing to use a behavior-change program might reasonably be expected to be motivated to change their depression. In contrast, participants recruited from the other entities listed above might have been more motivated by the financial compensation offered, rather than a desire to improve their depression symptoms. Further evidence of skewed motivations in this recruitment group is indicated in the very high numbers of individuals who were screened out by reporting much higher than expected rates of mania, and to a lesser extent, depressive symptoms during the online screening.Analyses of baseline data indicated that the non-EAP subjects had lower incomes, were less likely to be fully employed, and had less education. Since these three factors are also related to poorer outcomes in depression, these factors may at least partially explain why the program was not as effective for individuals with no EAP access ,92. WhilWe acknowledge several limitations in this initial efficacy trial and offer some caution in interpreting the findings. First, although random assignment was used, all the participants volunteered for the study and thus represent a convenience sample of interested individuals and cannot be considered representative of the general population. Second, participants completed self-report surveys, the validity and reliability of which may be somewhat suspect. Third, the reliability of some measures is only moderate and this may have attenuated the effect size of the intervention effects found in the study. Fourth, while the attrition rates in the study were relatively low, subjects were compensated for completing assessments. Thus, it cannot be concluded that the subject completion rate found here would occur at the same rate without compensation for participation. Lastly, attenuation of outcomes at 10-week follow-up suggests a need for more potent activation of CBT-based skills or a need for extended app contacts to drive continued engagement. Future research will explore the frequency and type of app contacts to optimize extended engagement and improve both short- and long-term efficacy of this light-touch, mobile approach to activating use of CBT skills to self-manage depressive symptomatology.Given the high prevalence of depression and the fact that most adults with depression never receive treatment, there is a critical need for effective interventions that can be widely and cost-effectively disseminated through multiple delivery channels. The MoodHacker mobile Web app has demonstrated potential for dissemination (1) as a self-guided intervention for individuals unwilling or unable to seek in-person depression services and (2) through EAP and similar health and wellness organizations supporting employed adults and their families. Ideally, exposure to MoodHacker needs to be extended beyond the 6-week time frame to maintain user engagement and improve longer-term efficacy. Further, the implementation and integration of MoodHacker within EAPs and similar organizations that can provide guidance from counselors seems likely to augment the effectiveness of the MoodHacker app.


Losses to follow-up (LTFU) remain an important programmatic challenge. While numerous patient-level factors have been associated with LTFU, less is known about facility-level factors. Data from the East African International epidemiologic Databases to Evaluate AIDS (EA-IeDEA) Consortium was used to identify facility-level factors associated with LTFU in Kenya, Tanzania and Uganda. Patients were defined as LTFU if they had no visit within 12 months of the study endpoint for pre-ART patients or 6 months for patients on ART. Adjusting for patient factors, shared frailty proportional hazard models were used to identify the facility-level factors associated with LTFU for the pre- and post-ART periods. Data from 77,362 patients and 29 facilities were analyzed. Median age at enrolment was 36.0 years , 63.9% were women and 58.3% initiated ART. Rates  of LTFU were 25.1 (24.7–25.6) and 16.7 (16.3–17.2) per 100 person-years in the pre-ART and post-ART periods, respectively. Facility-level factors associated with increased LTFU included secondary-level care, HIV RNA PCR turnaround time >14 days, and no onsite availability of CD4 testing. Increased LTFU was also observed when no nutritional supplements were provided (pre-ART only), when TB patients were treated within the HIV program (pre-ART only), and when the facility was open ≤4 mornings per week (ART only). Our findings suggest that facility-based strategies such as point of care laboratory testing and separate clinic spaces for TB patients may improve retention. The number of individuals accessing treatment for HIV has increased markedly over the last decade, particularly in sub-Saharan Africa (SSA) ,2. This Recently, the Joint United Nations Programme on HIV/AIDS (UNAIDS) have endorsed new global fast-track targets: 90% of all PLHIV to be diagnosed and know their status, 90% of all people diagnosed with HIV infection to receive ART, and 90% of those receiving ART to be virally suppressed by 2020 . RetentiVarious patient factors have been associated with becoming LTFU which include demographic  –13 and cThis retrospective cohort study involved patient-level and facility-level data from 29 sites affiliated with the East Africa (EA)-IeDEA Consortium. EA-IeDEA is a cohort of patients from HIV clinical care sites in Uganda, Kenya and Tanzania. Data are collected in the context of routine care at baseline and each follow-up visit, including socio-demographic data, the date of starting ART, type of treatment initiated, and, where available, CD4 counts and HIV-1 plasma RNA levels at baseline and during follow-up. All programs collect pre-ART data. Many programs may also collect contact information to facilitate patient tracing although not all programs trace patients who become LTFU. Tracing methods for missed visits vary between sites and may include mobile phone calls, SMS reminders, and/or home visits. Some clinics also involve volunteers from community-based organizations (CBO) to track patients.This analysis was limited to all adult patients, 14 years of age or older who were enrolled in a clinic associated with EA-IeDEA between May 2000 and May 2009. The exact date of database closure differed by program and ranged from 31 March 2008 to 19 May 2009. We excluded pregnant women from our analyses, mainly because of the variability in the frequency of follow-up for pregnant women and their movement between antenatal care (ANC) and HIV care which can affect retention. Indeed, previous studies have demonstrated higher rates of LTFU among pregnant women compared to non-pregnant women and other populations –18.In Kenya, participating programs included the United States Agency for International Development-Academic Model Providing Access to Healthcare (USAID-AMPATH), the Family AIDS Care and Education Services (FACES), and the MTCT (Mother-to-Child Transmission)-Plus program in Kisumu. In Uganda, affiliated sites included the Infectious Diseases Institute (IDI), Mbarara Immune Suppression Syndrome (ISS) clinic and the Makerere University-Johns Hopkins University (MU-JHU) collaboration at Mulago Hospital (MTCT-Plus program). Contributing sites in Tanzania included the Tumbi Regional Hospital in Kibaha, the Ocean Road Cancer Institute in Dar es Salaam, and the Morogoro Regional Hospital in Morogoro. Facility data came from a survey of all participating facilities conducted between August 2009 and February 2010 . All anaA socio-ecological framework was developed to guide our hypotheses about what measures to include in this analysis . In thisLoss to follow-up, the primary outcome, was defined as no clinic visits for at least 12 months for the pre-ART period and at least 6 months for the post-ART period, immediately prior to database closure. Six months has previously been proposed as the most informative definition of LTFU for individuals on ART . For inddemographic factors: age at enrolment (in years) and gender ; socio-economic characteristics: education level , disclosure of HIV status (yes/no), and travel time to clinic ; and clinical characteristics: on ART (yes/no), time on ART (in months), CD4 count at enrolment (cells/μl), CD4 count at ART initiation , World Health Organization (WHO) clinical stage at enrolment  and/or at ART initiation  (Stage 1–4), ever on TB treatment (yes/no) and calendar year at enrollment or ART initiation . Two periods were selected to account for the rapid expansion of highly active antiretroviral therapy in East Africa.Patient-level measures included General facility characteristics included location , HIV lab availability including HIV RNA PCR (on site vs. off site vs. not available) and CD4 count (on site vs. off site) as well as the turnaround time for HIV RNA PCR (≤14 days vs. >14 days) and CD4 count (≤7 days vs. >7 days). Availability of non- HIV labs (on site vs. off site) which included total lymphocytes, ALT/AST, and creatinine were also recorded as well as disruptions in CD4 reagents in the last year (no vs. yes). The level of care including whether the facility was considered primary , secondary  or tertiary  was also explored. Clinical operations included operating hours: open ≤4 mornings vs. >4 mornings during the week, open ≤ 4 evenings vs. >4 evenings during the week, and open on weekends (no vs. yes); presence of an electronic database to capture data on patients who transfer in (no vs. yes) and transfer out (no vs. yes); a waitlist for initiating ART (no vs. yes); daily average number of available physicians/mid-level staff (<3 vs. ≥3); ratio of adult patients to daily number of physicians/mid-level staff (<10 vs. ≥10); daily average number of lay outreach workers (<2 vs. ≥2); the ratio of adult patients to number of lay outreach workers (<10 vs. ≥10); whether cotrimoxazole prophylaxis, food rations/supplements, vitamins (including zinc), and nutritional treatment is provided (no vs. yes) and whether TB is diagnosed and treated within the ART program (yes vs. symptomatic patients referred to TB clinic). Retention strategies included whether pre-ART outreach services were available (no vs. yes), the number of days to track patients after a missed visit (≤7 days vs. >7 days), whether outreach workers phone missing patients first (no vs. yes), if a business number (no vs. yes) or a relative’s number (no vs. yes) was available, availability of home visits (no vs. yes), type of transport for home visits  and death ascertainment through active outreach (no vs. yes). While broader national contextual factors also influence LTFU, we were unable to measure such factors directly.Since little is known about how facility factors can influence retention in HIV care, we explored a range of available facility-level characteristics. We used our conceptual model to categorize facility factors of interest. Categorical variables were described using frequencies and proportions. Description of the quantitative variables was based on the median and interquartile range (IQR). Rates of LTFU were estimated using the maximum likelihood under an exponential distribution assumption, and the associated standard errors were calculated based on non-parametric bootstrap methodology. Modelling the cause-specific hazard of LTFU was based on the semi-parametric proportional hazards model, with a gamma-distributed shared frailty according to EA-IeDEA program to take into account the association between patients from the same EA-IeDEA program. Pre-ART and post-ART periods were analysed separately . Note thThe date of death was missing for 718 (13.7%) of the 5,256 deaths that occurred in this sample. In order to deal with missing death times, we performed hot-deck imputation  under thIn total, the analysis included 88,152 patients enrolled in one of the 29 clinics from 9 programs within the EA-IeDEA Consortium. The majority were female (68.3%) and 10,790 (18.0%) women were pregnant at some point during the follow-up period. After excluding pregnant women, the final sample size consisted of 77,362 patients.Demographic and clinical characteristics of the study sample are presented in A description of the key facility-level characteristics is given in Pre-ART and post-ART rates of LTFU by EA-IeDEA program are presented in Results from the multivariable analysis of LTFU both pre- and post-ART initiation are presented in While this analysis focused primarily on facility-level factors, various patient-level factors were also significant, and include younger age, more advanced HIV disease based on WHO stage, and lower educational level were all associated with increased hazards of LTFU. LTFU in the pre-ART follow-up period was more frequent among men and patients enrolled prior to 2007. Conversely, LTFU was slightly higher in the post-ART period among those initiating ART more recently (2007–2009 vs. 2000–2006). Tertiary care, the ability to identify patients who transferred in (electronic database), and having a waiting list for initiating ART were not significantly associated with LTFU in the pre-ART period. The provision of nutritional supplements, having a waiting list for initiating ART, referring TB symptomatic patients to a TB clinic, phone first for outreach, and being male were not significantly associated with LTFU from ART.In this comprehensive analysis of facility-level factors associated with LTFU in 77,362 patients from 29 facilities across three east African countries, we found numerous facility-level factors that were associated with reduced LTFU: facility open more than 4 mornings per week; provision of care at secondary-level facilities such as district hospitals compared to primary care facilities; provision of nutritional supplements, provision of onsite laboratory services and faster CD4 results; and provision of TB care separate from HIV and ART care. These findings suggest some specific modifiable facility-level strategies that programs and facilities should consider when planning and implementing their services to promote retention in HIV care and in turn, maintenance of viral suppression. We found that LTFU rates were also higher in the pre-ART period compared to the ART period, although this is likely to reduce with progressive adoption and implementation of a “treat all” strategy as recommended by WHO, minimising time between diagnosis and ART initiation .LTFU rates were lower in secondary-level facilities compared to primary care facilities. Other studies have shown that decentralization is associated with improved retention , 24, as Facilities that were open more than 4 mornings per week reported fewer losses to follow-up. More available and flexible opening hours , 27 may Provision of onsite laboratory services and faster results turnaround time was associated with reduced LTFU. Our finding largely relates to delays in CD4 testing rather than viral load , and also may serve as a proxy for facility function and responsiveness. It is recognized that limited human resources, lack of timely CD4 testing (due to broken CD4 machines or missing reagents)  and longFinally, our study found that management of TB within the HIV and ART program rather than in a separate TB programme was associated with a higher hazard of LTFU. HIV-TB co-infection remains a major problem in sub-Saharan Africa and continues to present numerous challenges to HIV treatment programs, particularly in infection control and risks of TB transmission and acquisition in an HIV infected population . In respThere were several limitations to the present study. The facility-level data available were based on a site assessment that was conducted in late 2009 while the patient-level data was collected from 2000 through early 2009. However, we consider that the critical patient and service delivery factors still apply, and an updated assessment is planned to identify the impact of more recent changes in facility-level factors on retention. There were also several potentially important factors reported in other studies that we did not capture—these include the impact of patient-provider relationships, clinic visit wait times, and transport costs to the clinic –15, 24, Overall our findings support the implementation of several key strategies to improve retention in care: onsite and timely laboratory testing; flexible facility hours and daily clinics, provision of care in secondary level health facilities that can provide a more comprehensive range of services. This will become more critical with continued scale up of ART to all 34 million persons living with HIV. This is particularly the case in light of the increasing burden of co-morbidities, such as tuberculosis, diabetes, or hypertension, and given the move towards more comprehensive reproductive health care to prevent maternal-child HIV transmission. If we are to meet the UNAIDS 2020 targets and fully realize the individual and public health benefits of ART, health care facilities must be both evidence-based and patient –centered, along each stage of the care-prevention continuum.S1 File(DOC)Click here for additional data file.


Colorectal cancer (CRC) is the second leading cause of cancer-related death in developed countries. Early detection of CRC leads to decreased CRC mortality. A blood-based CRC screening test is highly desirable due to limited invasiveness and high acceptance rate among patients compared to currently used fecal occult blood testing and colonoscopy. Here we describe the discovery and validation of a 29-gene panel in peripheral blood mononuclear cells (PBMC) for the detection of CRC and adenomatous polyps (AP). Blood samples were prospectively collected from a multicenter, case-control clinical study. First, we profiled 93 samples with 667 candidate and 3 reference genes by high throughput real-time PCR (OpenArray system). After analysis, 160 genes were retained and tested again on 51 additional samples. Low expressed and unstable genes were discarded resulting in a final dataset of 144 samples profiled with 140 genes. To define which genes, alone or in combinations had the highest potential to discriminate AP and/or CRC from controls, data were analyzed by a combination of univariate and multivariate methods. A list of 29 potentially discriminant genes was compiled and evaluated for its predictive accuracy by penalized logistic regression and bootstrap. This method discriminated AP >1cm and CRC from controls with a sensitivity of 59% and 75%, respectively, with 91% specificity. The behavior of the 29-gene panel was validated with a LightCycler 480 real-time PCR platform, commonly adopted by clinical laboratories. In this work we identified a 29-gene panel expressed in PBMC that can be used for developing a novel minimally-invasive test for accurate detection of AP and CRC using a standard real-time PCR platform. Colorectal cancer (CRC) is the third most common cancer and second leading cause of cancer-related death among men and women in Europe . ImportaColonoscopy is the “gold standard” for AP and CRC diagnosis, however it is not the preferred method for mass screening because of its cost, invasiveness, low compliance and limited accessibility. Currently recommended non-invasive methods for mass screening include immunochemical and guaiac fecal occult blood testing . Yet, compliance with fecal tests is still suboptimal in countries with an FOBT screening program , 5, 6. TWhen searching for differences in gene expression and identification of RNA transcripts to be subsequently used as potential biomarkers, commonly used methods include microarray-based DNA hybridization platforms or RNA-based sequencing techniques , 19. AltHere we report the discovery and characterization of a 29-gene panel in PBMC for the detection of colorectal adenomas and carcinomas using a nanoliter high throughput qPCR platform (OpenArray) . To thisA case-control study (DGNP-COL-0310), including three South Korean and six Swiss centers which enrolled 1665 subjects older than 50 years that were referred for colonoscopy by general practitioner or were scheduled for surgery, was conducted from June 2010 to April 2013. The study was specifically conceived and designed for the development and validation of a new test for CRC screening. The biomarker discovery phase took place during the first half of patient recruitment. For this purpose, a subset of 144 subjects, allocated to control, CRC and AP groups , was ranSubjects had no first-degree family history of CRC or a known CRC predisposition, previous history of polyps or cancer including CRC, no hepatobiliary, genitourinary, autoimmune and inflammatory disorders, including inflammatory bowel diseases, infectious diseases and fever within 4 weeks before colonoscopy. Chronic diseases common in the old population, such as diabetes, hypertension, hypercholesterolemia, heart failure, were not considered as exclusion criteria. A control subject was defined as an individual without any past and present history of colorectal lesions or diseases . The AP group included subjects diagnosed with an adenoma larger than 1 cm, based on the endoscopic measurement. The CRC group included patients with carcinoma at all four TNM stages. Final diagnosis was based on colonoscopy and histopathological evaluation.The study protocol (DGNP-COL-0310) was approved by the competent review boards and ethics committees for research on human subjects of Canton Bern, Switzerland , Canton St. Gallen, Switzerland , Canton Vaud, Switzerland , Canton Basel, Switzerland , of the Severance Hospital, Yonsei University College of Medicine, South Korea (No. 4-2010-0128) and by the Institutional Bioethics Review Board of Seoul National University Hospital, South Korea (IRB No. H-1004-020-315). Written informed consent was obtained from all study participants adhering to the local ethical guidelines.later Solution  and stored at -80°C.Peripheral blood from all subjects was drawn either up to 30 days before or up to 12 weeks after colonoscopy and prior to any polyp or cancer resection or pre-operative chemotherapy. Blood samples were collected into 4x4 ml BD Vacutainer CPT tubes . Filled CPT tubes were kept at room temperature and PBMC separation performed within 6 hours according to manufacturer’s instructions. PBMC pellets were resuspended in RNAAutomated purification of total RNA was performed on QIAcube by RNeasy Mini kit  and included a DNase treatment. RNA concentration was measured by Nanodrop spectrophotometer  and RNA integrity was analyzed by Agilent 2100 Bioanalyzer . Samples with a RIN < 7 were considered of poor quality and discarded. On average RNA showed a RIN of 9±0.5. Isolated total RNA was aliquoted and stored at -80°C. In order to meet the high RNA concentration required by the RT protocol, RNA samples were systematically precipitated following a standard 100% ethanol/3M sodium acetate method.In order to find relevant blood biomarkers for CRC, we performed gene expression screening on 144 samples derived from patients with AP or CRC and control subjects. The screening was conceived in 2 phases . First, i.e. control versus CRC or control versus AP): a p-value less than 0.1 or a fold-change greater than 1.5 . In addition, biomarkers previously identified in our laboratory [vs. 168-assay format). The expression levels obtained in both phases for these 40 samples were highly correlated (R2 = 0.993)  and a median SD of 0.7 (inter-quartile range: 0.59–1.04) . Gene pr= 0.993) , prompti= 0.993) . To compNine hundred ng of total RNA was reverse transcribed in 20 μl volume using the high-Capacity cDNA Reverse Transcription kit  with random primers, according to the manufacturer’s instructions.Gene expression profiling was performed using the OpenArray system   according to manufacturer’s instructions.nd derivative maximum method [RealTime ready Custom RT-qPCR assays  based on Universal ProbeLibrary (UPL) technology , were prm method .PCR data derived from the OpenArray and the LC480 platforms were normalized by the ΔCT method using the mean of three housekeeping genes RPLP0, NACA and TPT1. These genes were selected because they were the most stable in 3 PBMC-related microarray dataset available from the GEO database  and alsoGene expression fold change, was defined as Wilcoxon rank test  was applThe multivariate analysis used for feature selection in phase 2 screening included the following methods: K-top scoring pair, a parameter-free, feature selection algorithm , and penTo evaluate the predictive accuracy of the 29-gene panel, penalized logistic regression models were fitted on the dataset and validated by non-overlapped bootstrap method [The Pearson correlation coefficient was used to evaluate linear correlation between genes’ measurements by two instruments.The R statistics environment was used for statistical analyses.The dataset generated from phase 2 screening (163 genes and 144 samples) was analyzed and filtered for low expression and unstable genes across the two phases, and 20 genes were further discarded, reducing the number of candidate genes to 140. The data were explored in order to define which genes, alone or in combinations, had the highest power to discriminate CRC, AP and AP together with early stage CRC (AP+CRC I-II) from the control group . In addiMultivariate analysis was applied to the dataset to discriminate CRC, AP, AP+CRC from control group. It included KTS-pair  and fiveThe univariate and multivariate gene lists were then merged, resulting in a final list of 29 genes . Intereswww.qiagen.com/ingenuity), revealed that the panel was enriched in genes involved in leukocytes migration and chemotaxis   . These cTo evaluate the clinical relevance of our 29-gene panel, in particular its predictive accuracy, penalized logistic regression was applied to the dataset and fitted models were validated by non-overlapped bootstrap method. Models could discriminate CRC or AP >1cm from controls with an average sensitivity of 75% and 59%, respectively. The average specificity, defined as the number of controls correctly classified over the total number of controls, was 91%. ROC analysis determined an AUC of 0.88  and of 0.85  for CRC or AP detection, respectively . When thThe OpenArray platform demonstrated to be valuable for high throughput gene expression profiling and biomarker discovery. However, this platform is not suitable in a routine clinical laboratory setting, in which easiness of use, flexibility and low costs are preferred. With the aim of developing the 29-gene panel into a widely used CRC screening test, we evaluated the panel behavior on a qPCR platform that is commonly adopted by clinical laboratories. The 144 samples were profiled with the 29-gene panel using a LightCycler 480 (LC480) instrument and a set of commercially available probe-based assays , preloaded on 384-well plates.Correlation and linear regression analysis showed that gene expression levels measured on the two platforms were highly comparable (correlation coefficient: 0.933) . In geneIn this work we have identified a 29-gene panel expressed in PBMC, capable of discriminating individuals with AP >1cm or CRC from healthy individuals. Penalized logistic regression analysis correctly classified 75%, 59% (sensitivity) and 91% (specificity) of CRC, AP and controls, respectively.+ cells mobilized from the bone marrow in response to the growing primary tumor, thereby generating pro-angiogenic or pro-metastatic CD11b+ cells, respectively [The approach we used is different compared to existing screening tests for CRC, as it is based on PBMC genes expression profiles. This leverages the well-established concept of tumor-host interaction and contribution of bone marrow-derived cells to tumor progression , 17. It ectively , 37.We recognize that genes associated to inflammatory processes might be similarly modulated in inflammatory conditions/diseases other than CRC or AP, possibly leading to reduced signature specificity. However, the weight of possibly “non-specific” genes is moderated by the information carried by other genes in the panel. We are planning to evaluate the gene panel in an independent test set of samples collected during this clinical study, including several inflammatory conditions, in particular inflammatory bowel diseases and to use this sample set to optimize a predictive algorithm highly specific for CRC and AP detection.Technically, our discovery strategy leveraged the recent development of accurate and sensitive high-throughput qPCR platforms based on microfluidic technologies, such as the OpenArray . The appThe findings reported here are in line with the recently reported results obtained from a pilot monocentric study in which the feasibility of the use of a PBMC-derived signature to detect CRC and AP was demonstrated . The siget al. but not significant in phase 1. Moreover, the predictive accuracy reported in the two studies is very similar, thereby demonstrating the consistency and robustness of our approach. The fact that the two panels overlap only for a fraction of genes could be explained by the fact that the biomarker discovery reported by Nichita et al. focused predominantly on adenoma samples rather than CRC. However, it is already known that different gene signatures may carry the same biological information as it was reported in gene-expression signatures in breast cancer [In spite of the major clinical and technical differences in these studies, 8 genes were found common to the 29-gene panel reported here and the 42-gene panel previously reported [t cancer .In conclusion, we have discovered and characterized a 29-gene panel in PBMC for the detection of colorectal adenomas and CRC. The signature can discriminate AP >1cm and CRC from controls with an average sensitivity of 59% and 75%, respectively, and a specificity of 91%. We also demonstrated that the gene panel could be easily transferred and implemented into a medical laboratory-friendly assay, which is a key step in the development of a new cost-effective, simple blood-based colorectal cancer screening test. The identified signature will be the basis for developing a decisional algorithm that will be validated for its prospective discriminatory value on the remaining samples collected in this case-control multicenter study. The availability of a larger number of AP>1 cm and early stage CRC samples, as well as the presence of patients with inflammatory diseases or other type of tumors, will allow to finely tune the algorithm to be highly sensitive and specific to precancerous and early stage cancerous lesion.S1 Fig2 = 0.993).Forty samples were analyzed in both phase 1 and phase 2 and Ct values compared. Ct1 refers to Ct values obtained in the phase 1 and Ct2 in the phase 2. Mean Ct values obtained in the two phases were highly correlated (R(TIFF)Click here for additional data file.S1 TableFor each of the gene is reported the official gene symbol, the full gene name, the RNA reference sequence database ID (RefSeq), the TaqMan assay ID  used during the screening as well as, if any, the alternative assay used in phase 2 (TaqMan assay 2), and at last the genes selected for phase 2 screening.(XLSX)Click here for additional data file.S2 TableIn this phase 93 PBMC samples, equally distributed among controls (Con), adenomatous polyps >1cm (AP) and CRCs stage I-IV , were profiled with 670 genes. The table displays only those 534 genes showing expression levels above the limit of detection for more than 50% of the samples or a good PCR amplification. Mean, standard deviation (SD) and median values are reported for four main analysis groups ) and for two subgroups: CRC stages I-II only (CRCI-II) and AP plus CRCI-II (AP-CRCI-II). During the univariate analysis, the disease groups/subgroups were compared to the control one and the AP group was evaluated against the CRC one. For each gene and for each group analysis, p-values derived from Student’s t-test and Wilcoxon rank sum test and fold-change (FC) linear values, derived from the mean or median, are reported. In addition, the table indicates if the gene was selected for the phase 2 screening (163 genes) and in the final 29-gene panel.(XLSX)Click here for additional data file.S3 TableIn this phase 144 PBMCs samples, including 50 controls (Con), 46 adenomatous polyps >1cm (AP) and 48 CRCs stage I-IV , were profiled with 163 genes. Univariate and multivariate analysis were conducted only on the 140 genes showing stable and reproducible measurements between the two phases. For the univariate analysis, the same settings explained in (XLSX)Click here for additional data file.S4 TableRealTime ready Custom RT-qPCR assays  used to validate the gene panel on the LightCycler 480 instrument are reported, including forward and reverse primer sequences and the associated UPL probe ID. The assays were pre-loaded on 384-well plates. Reference genes used for PCR values normalization are marked with *.(PDF)Click here for additional data file.


Antiretroviral therapy (ART) is crucial for controlling HIV-1 infection through wide-scale treatment as prevention and pre-exposure prophylaxis (PrEP). Potent tenofovir disoproxil fumarate-containing regimens are increasingly used to treat and prevent HIV, although few data exist for frequency and risk factors of acquired drug resistance in regions hardest hit by the HIV pandemic. We aimed to do a global assessment of drug resistance after virological failure with first-line tenofovir-containing ART.RT) gene.The TenoRes collaboration comprises adult HIV treatment cohorts and clinical trials of HIV drug resistance testing in Europe, Latin and North America, sub-Saharan Africa, and Asia. We extracted and harmonised data for patients undergoing genotypic resistance testing after virological failure with a first-line regimen containing tenofovir plus a cytosine analogue (lamivudine or emtricitabine) plus a non-nucleotide reverse-transcriptase inhibitor . We used an individual participant-level meta-analysis and multiple logistic regression to identify covariates associated with drug resistance. Our primary outcome was tenofovir resistance, defined as presence of K65R/N or K70E/G/Q mutations in the reverse transcriptase . Pre-ART CD4 cell count was the covariate most strongly associated with the development of tenofovir resistance . Use of lamivudine versus emtricitabine increased the risk of tenofovir resistance across regions . Of 700 individuals with tenofovir resistance, 578 (83%) had cytosine analogue resistance (M184V/I mutation), 543 (78%) had major NNRTI resistance, and 457 (65%) had both. The mean plasma viral load at virological failure was similar in individuals with and without tenofovir resistance .We recorded drug resistance in a high proportion of patients after virological failure on a tenofovir-containing first-line regimen across low-income and middle-income regions. Effective surveillance for transmission of drug resistance is crucial.The Wellcome Trust. More than 35 million people worldwide are living with HIV-1.The global scale-up of cART has now reached 15 million treated individuals.RT) gene.In individuals receiving tenofovir, HIV-1 develops phenotypically and clinically significant resistance usually as a result of one mutation at position 65  in the reverse transcriptase  in patients with HIV-1, published between January, 1999, and June, 2015, using the search terms “HIV” AND “tenofovir” AND “resistance”. We identified studies done in untreated adults (age >15 years) in which either efavirenz or nevirapine was combined with tenofovir and either emtricitabine or lamivudine as first line antiretroviral therapy. Several studies reported resistance data for tenofovir when the drug was started after initial use of stavudine or zidovudine; these studies were not reviewed further. We also excluded studies that reported tenofovir use without NNRTI because standard first-line antiretroviral therapy under a public health approach is based on NNRTI in adults.We identified randomised controlled trials and a meta-analysis comparing NNRTI with protease inhibitors, in combination with tenofovir, which reported resistance data. Patients in high-income settings reported tenofovir resistance in 0–25% of virological failures and those in sub-Saharan Africa in 28–50%. The only other prospective study in sub-Saharan Africa was PASER-M, and was limited by few resistance data for patients given tenofovir plus NNRTI-based combination antiretroviral therapy (cART). The remaining studies were largely from South Africa and reported a wide range of prevalence (between 23% and 70%) of tenofovir resistance after virological failure. In west Africa, one study reported that 57% of virological failures were tenofovir resistant in a very small sample of 23 patients. Although aforementioned studies also reported NNRTI and cytosine analogue resistance, they were unable to quantify to what extent tenofovir resistance was a marker for high-level compromise of the regimen. We found no studies that specifically reported resistance data for patients given first-line tenofovir in east Africa. No study reported resistance data from more than one continent, and none seemed adequately powered to establish the effect of co-administered reverse-transcriptase inhibitors on the emergence of tenofovir resistance.Added value of this studyThis study reports the most comprehensive assessment of HIV-1 drug resistance after scale-up of first-line WHO recommended tenofovir-based antiretroviral regimens, showing that tenofovir resistance is surprisingly common in patients with treatment failure across many studies in all low-income regions. Importantly, these individuals also have notable resistance to other drugs in their regimen, leading to almost complete compromise of combination treatment. Challenging current perceptions in the specialty, our findings show that tenofovir resistant viruses have substantial transmission potential. Furthermore, our results show that viral strain affects tenofovir resistance in Europe but is not the main driver for resistance in viruses circulating in sub-Saharan Africa. Newly identified risk factors for resistance to tenofovir and NNRTI drugs include pre-treatment CD4 cell count  and co-administered antiretrovirals.Implications of all the available evidenceImprovements in the quality of HIV care and viral load monitoring could mitigate the emergence and spread of tenofovir resistance, thereby prolonging the lifetime of tenofovir-containing regimens for both treatment and prophylaxis. Surveillance of tenofovir and NNRTI resistance should be a priority both in untreated and treated populations.The TenoRes collaboration comprises adult HIV treatment cohorts and clinical trials from Europe, Latin and North America, sub-Saharan Africa, and Asia. Cohorts and trials were identified by RWS and RKG as those known to do genotypic resistance testing through previous collaborations, the WHO HIV Drug Resistance Network, and through the International HIV Drug Resistance Workshop. Moreover, we did a systematic review using the keywords “HIV”, AND “tenofovir” AND “resistance” in PubMed for articles published between January, 1999, and June, 2015. We identified 44 studies suitable for the reported analysis after applying the following inclusion criteria: documented virological failure after first-line ART comprising tenofovir plus either lamivudine or emtricitabine plus either efavirenz or nevirapine ; a successful resistance test result associated with virological failure of cART; tenofovir-based ART for at least 4 months before virological failure; and absence of thymidine analogue mutations at resistance testing . ExclusiStanford HIV Drug Resistance Database.We collected individual-level data for a predefined set of covariates: age at first-line ART initiation, sex, frequency of viral load monitoring (number of tests per year), urban versus rural setting for HIV clinics, viral load threshold for virological failure and genotyping, co-administered antiretrovirals, duration of treatment, viral load and CD4 cell count before the start of first-line ART (baseline) and at time of viral failure, and resistance mutations based on the vs ≥100 cells per μL), baseline viral load , defined as specific mutations at aminoacid positions 100, 103, 106, 108, 181, 188, 190, and 225,We did sensitivity analyses to investigate whether associations changed when adjusted for possible confounders. Because of the sparseness of data in many studies, we were unable to adjust within-study associations for potential confounders. Instead, we did additional analyses using logistic regression models with a random effect at study level to estimate associations before and after adjustment for possible confounders in a common subset of participants. To build the adjusted model, we included each of our main exposures and HIV subtype. We also considered for inclusion individual-level information about age, sex, year of treatment initiation, and length of time on tenofovir, but rejected these covariates because of a lack of any univariate association with tenofovir resistance. We chose to only use these models for working out the likely extent of confounding, because estimated associations from these models are partly derived from between-study comparisons.To clarify whether the association between baseline CD4 or baseline viral load and tenofovir resistance was linear , we categorised participants into four categories based on baseline CD4 cell count  or baseline viral load . We assessed associations by plotting the estimated OR against the mean level of baseline CD4 , in a random-effects logistic regression model adjusted for region, co-administered drugs, and baseline viral load (or baseline CD4).To assess the potential transmissibility of mutant viruses, we graphically compared the distribution of plasma HIV-1 RNA concentrations of patients from the same study with and without tenofovir resistance.We did not use multiple imputation to adjust for missing data because most missing data were the result of a lack of availability at the study level. Instead, we restricted analyses to the subset of participants with information available about all relevant covariates for each specific analysis. The The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. RKG and JG had full access to all the data in the study and had final responsibility for the decision to submit for publication.The TenoRes collaboration included 1926 individuals from 36 countries . Table 1vs lamivudine) and efavirenz (vs nevirapine) varied significantly by region. Emtricitabine was used significantly more than lamivudine in Europe, North America, and west and central Africa, and efavirenz was used significantly more than nevirapine in all regions apart from east and west and central Africa. The median duration of ART ranged from 11 to 26 months. Pre-treatment viral load ranged between 4·80 and 5·58 log copies per mL and was significantly higher in eastern and western and central Africa and Latin America than the other regions  and multivariate logistic regression analyses . We notevs 133 900 copies [SE 16 650]; p=0·626; Finally, we compared the viral load at treatment failure in the presence and absence of tenofovir-associated mutations. The mean plasma viral load at treatment failure was not different in the presence or absence of tenofovir associated mutations  of patients in sub-Saharan Africa have virological failure by 12 months.There are several important limitations of our study. First, because we only included patients with virological failure related to existing study cohorts,Third, our findings on risk factors for tenofovir resistance were derived from an unadjusted meta-analysis involving very different study populations. Although this enhances the generalisability of results, it has the potential to lead to biased comparisons. However, we took measures to minimise biases. We exclusively used within-study and within-country comparisons for our primary analyses, thereby ensuring that comparisons were for participants undergoing similar treatment monitoring practices. We tested associations between risk factors and found that they were generally weak. For example, baseline CD4 cell count and viral load were only weakly associated with one another and neither was strongly associated with type of co-administered drug. Additionally, we undertook sensitivity analyses, which suggested that adjustment for other covariates had minimum effect on estimated associations. Lastly, our data tended to be consistent with previous studies—eg, our findings of higher resistance in subtype C patients are consistent with in-vitro data suggesting subtype C viruses are more susceptible to developing the K65R mutation.Fourth, despite our analysis being the largest drug resistance study ever undertaken after failure of first-line tenofovir-containing cART, patient numbers were somewhat limited by the slow uptake of tenofovir-based regimens in west and central Africa, eastern Europe, and Asia (in particular China and Russia), and information about baseline viral load in these settings was uncommon. As a result, European countries, Thailand, and South Africa contributed substantially to the analysis.In summary, extensive drug resistance emerges in a high proportion of patients after virological failure on a tenofovir-containing first-line regimen across low-income and middle-income regions. Optimisation of treatment programmes and effective surveillance for transmission of drug resistance is therefore crucial.ravindra.gupta@ucl.ac.ukCorrespondence to: Dr Ravindra K Gupta, UCL, Department of Infection, London WC1E 6BT, UK orrshafer@stanford.edurProf Robert W Shafer, Department of Medicine, Stanford University, Stanford, CA 94305, USA


The demographic transition and increasing life expectancy in Africa has lead to a rising elderly population. In Nigeria, little is known about the profile of and treatment outcomes of tuberculosis (TB) in the elderly.Retrospective cohort study of adult TB patients treated between January 2011 and December 2012 in two large health facilities in Nigeria. The demographic, clinical and treatment outcomes of patients aged 60 and older were compared with those aged 15 to 59 years.Elderly (≥60 years) TB patients accounted for 12.7% of all (1668) adult TB enrolled. Elderly patients had a higher proportion of men compared to non-elderly ; but a lower proportion of smear-positive TB at baseline . A higher proportion of elderly patients failed to smear convert after the intensive phase of treatment , and overall elderly patients had lower treatment success rates . Unsuccessful outcomes were mainly due to higher default and deaths in the elderly. The risk factors for unsuccessful outcomes in the elderly were: extrapulmonary TB case (adjusted odds ratio (aOR) 10.9; 95% confidence interval (CI) 1.1–108), and HIV co-infection .Treatment outcomes of elderly TB patients were inferior to non-elderly adults with higher death and default rates being implicated. With the rising elderly population, specific strategies are needed to quickly address TB management in the elderly in resource-limited settings. Globally, the frequency of tuberculosis among elderly persons is about three times that observed in non-elderly adults With the current demographic transition and increasing life expectancy in low- and middle-income countries The aim of this study was to describe the epidemiological characteristics and treatment outcomes of elderly patients with tuberculosis in a high-incidence African setting where patients were managed within a TB control programme with significant resource limitations. Specific objectives were to determine among adult TB patients in Nigeria in 2011 and 2012: (i) the proportion of elderly (≥60 years) persons with TB and compare them with non-elderly adult TB cases in terms of their demographic and clinical characteristics, (ii) the differences in the treatment outcomes of elderly and non-elderly TB cases stratified by: gender, type of TB, TB regimen received, residence, treatment category, HIV status and facility where care was given, and (iii) the determinants of unsuccessful treatment outcomes among elderly TB patients.The study complies with international guidelines for research and was approved by the Ethics and Research Advisory Committee of the National Tuberculosis Control Programme, Ministry of Health, Ebonyi State, Nigeria. As this was a retrospective study, the consent of the patients was not obtained; however, patient records was anonymized and de-identified prior to analysis.This was a retrospective cohort study using routine programme data from two hospitals in the Nigerian state of Ebonyi. It included all elderly and non-elderly adult TB patients registered for treatment between 1 January 2011 and 31 December 2012.The study was conducted in one rural faith-based (private) secondary care hospital and the only tertiary (public) hospital in Ebonyi State, Southeastern Nigeria. Ebonyi state is one of Nigeria's 36 states; it has an estimated population of 2.5 million people Persons who developed a cough lasting two or more weeks with or without night sweats, fever, weight loss, shortness of breath, and sputum production were evaluated, registered and treated according to the WHO/NTP guidelines nd month of treatment for new cases or 3rd month of treatment for re-treatment cases  or 4th month (re-treatment cases) of treatment before commencing the continuation phase) Treatment follow-up was done using clinical and laboratory evaluations at intervals according to standard methods The study included all elderly TB patients (≥60 years) and non-elderly adult TB patients (15–59 years) registered for treatment at the two study hospitals between 1 January 2011 and 31 December 2012.Treatment outcomes were defined in accordance with standard WHO and NTP definitions as follows Data were sourced from the TB treatment registers of the study sites, were checked for errors, double-entered, and analyzed using Epi Info 3.4.1 . The following variables were collected: TB registration number, age, gender , treatment category (new vs. retreatment case), residence , type of TB (pulmonary vs. extrapulmonary TB), human immunodeficiency virus (HIV) status (HIV-positive vs. HIV-negative), health facility where care was given (private vs. public) treatment regimen received (6-month vs. 8-month regimen), sputum positivity before starting treatment and at the end of the intensive phase, and treatment outcomes.P<0.25 were included in the multivariable model. A backward elimination approach was used to find the best model. P<0.05 were considered statistically significant.Categorical group comparisons between elderly and non-elderly patients were made using the Chi-square test. Relative differences in rates of unsuccessful outcomes between these two groups were compared using crude odds ratios (ORs) and adjusted ORs. Adjusted ORs were determined through multivariable logistic regression analysis. We performed a stratified analysis to determine the occurrence of interaction and confounding between the outcome variable and the explanatory variables. All variables that were of clinical importance and all with a univariate Of 1668 TB patients comprising of 1574 pulmonary TB (985 smear-positive and 589 smear-negative cases) and 94 extrapulmonary TB enrolled; 212 (12.7%) were elderly (≥60 years) and 1456 (87.3%) were non-elderly. The mean (SD) age of the elderly patients was 66.7 (6.5) years. Demographic and clinical characteristics of elderly and non-elderly patients are shown in p = 0.06).Only 929 (94.4%) of the smear-positive TB patients had a sputum smear microscopy done after the intensive phase of treatment i.e. 853 non-elderly and 76 elderly. Of the elderly patients, 18/76 (23.7%) failed to smear convert after the intensive phase of treatment compared to 169/853 (19.8%) of the non-elderly patients (p = 0.01), higher in elderly patients who received care at the private health facility , higher in elderly new patients , and higher in elderly patients with pulmonary TB . Irrespective of HIV status, rates of unsuccessful outcomes were significantly higher in elderly than non-elderly patients . In patients who received the longer (8-month) anti-TB regimen there was no difference in the rates of unsuccessful outcomes between elderly and non-elderly patients (p = 0.52). However, among those who received the 6-month anti-TB regimen rates of unsuccessful outcomes were higher in elderly compared to non-elderly patients  (p = 0.2).The treatment outcomes of elderly and non-elderly TB patients stratified by their demographic and clinical characteristics are as shown in = 0.001) . Also, eUnivariate and multivariable logistic regression analysis was performed to determine socio-demographic and clinical risk factors for unsuccessful outcomes among elderly patients . The indTB in the elderly is emerging as a major global health issue globally and the control of TB in this group is crucial for a successful TB control programme The proportion of TB occurring in elderly patients in this study (12.7%) is comparable to the findings of studies in Senegal where the prevalence was 12.8% Compared with non-elderly patients, elderly individuals were less likely to be HIV-infected in this study. This agrees with a previous study Consistent with findings from Germany, India, Mexico and Taiwan, we have shown that smear-positive microscopy results were seen less frequently in the elderly The negative smear conversion of smear positive TB cases is an important determinant of treatment success Treatment success rate were lower and unsuccessful treatment outcomes, mainly death and default, were more frequent in the elderly than non-elderly patients in this study. This supports the finding of other studies in Africa, Asia, North America, and Europe This study showed that treatment success rates of elderly were inferior to that of non-elderly patients who were rural residents, had extrapulmonary TB, treated in a private facility, received the current 6-month anti-TB regimen as well as those with or without HIV infection. In order to better understand these differences in treatment success rates, we assessed the determinants of unsuccessful outcomes in elderly patients. Elderly patients who received the 6-month regimen had higher proportions of unsuccessful outcomes than those given the 8-month regimen. Although the crude and adjusted odds ratios did not reach statistical significance, this study suggests the need to further evaluate the effect of adjusting the duration of the current 6-month anti-TB regimen on treatment outcomes among elderly TB patients in resource poor settings. HIV co-infection and presence of extrapulmonary TB were independent predictors for unsuccessful outcomes in the elderly – these factors are not specific to the elderly patients alone There are several limitations to this study. The study was based on routine surveillance data. We were not able to assess for relationships between TB treatment outcomes in the elderly and other important variables like diabetes mellitus, renal failure, and other co-morbidities. Also, as TB/HIV collaborative activities were recently expanded in Ebonyi state, data on HIV status of all patients were available; but data on those who were started on antiretroviral therapy were not adequately captured in the tuberculosis treatment registers. Thus, other data like details of antiretroviral therapy use, cotrimoxazole prophylaxis and CD4+ count were not available for analysis. Furthermore, there were no information on the duration of symptoms before treatment, chest radiography, toxicity of anti-TB drugs, or causes of death; these variables may affect treatment outcomes.In conclusion, this study has shown that TB in the elderly occurred more frequently in men; they are less likely to have smear positive disease, and less likely to show negative smear conversion after the intensive phase of treatment. Also, compared with non-elderly TB cases, elderly TB patients are more likely to have unsuccessful treatment outcomes. The findings of this study have a number of practice and policy implications. We recommend that: 1) special interventions to reduce treatment default and mortality in elderly patients with TB needs to be implemented; 2) specific strategies to reduce unsuccessful outcomes among elderly patients in the identified high-risk groups should be implemented; 3) expansion of case finding and treatment of HIV among elderly TB patients should be improved; 4) Further studies on the impact of the current 6-month regimen on TB treatment outcomes in the elderly needs to be carried out and its relationships with the duration of treatment assessed; and 5) TB treatment registers in Nigeria and elsewhere should be modified to collect additional data such as co-morbidities, adverse drug reactions, and concomitant therapies from TB patients treated.Table S1Dataset on profile and treatment outcomes of tuberculosis in the elderly in Southeastern Nigeria, 2011–2012.(MDB)Click here for additional data file.


Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinomas (OPSCCs) have an improved prognosis compared to HPV-negative OPSCCs. Several theories have been proposed to explain this relatively good prognosis. One hypothesis is a difference in immune response. In this study, we compared tumor-infiltrating CD3+, CD4+, CD8+ T-cells, and granzyme inhibitors  between HPV-positive and HPV-negative tumors and the relation with survival.Protein expression of tumor-infiltrating lymphocytes (TILs)  and granzyme inhibitors was analyzed in 262 OPSCCs by immunohistochemistry (IHC). Most patients (67 %) received primary radiotherapy with or without chemotherapy. Cox regression analysis was carried out to compare overall survival (OS) of patients with low and high TIL infiltration and expression of granzyme inhibitors.p < 0.001) compared to HPV-negative OPSCCs. A high level of CD3+ TILs was correlated with a favorable outcome in the total cohort and in HPV-positive OPSCCs, while it reached no significance in HPV-negative OPSCCs. There was expression of all three granzyme inhibitors in OPSCCs. No differences in expression were found between HPV-positive and HPV-negative OPSCCs. Within the group of HPV-positive tumors, a high expression of SERPINB1 was associated with a significantly worse overall survival.HPV-positive OPSCCs were significantly more heavily infiltrated by TILs (HPV-positive OPSCCs with a low count of CD3+ TILs or high expression of SERPINB1 have a worse OS, comparable with HPV-negative OPSCCs. This suggests that the immune system plays an important role in the carcinogenesis of the virally induced oropharynx tumors. The causative role of the human papillomavirus (HPV), especially in oropharynx squamous cell carcinoma (OPSCC), is widely established . StudiesThe exact cause of this survival advantage is not fully elucidated and seems to be related to multiple factors such as the absence of field cancerization and wild-type expression of p53 and Rb . AdditioFor many cancers, it has become clear that in addition to genetic and epigenetic changes in the neoplastic cells, also microenvironmental factors play an important role in carcinogenesis and tumor progression. Cancers can survive the host immune response by escaping recognition and suppression of the host immune system . Indeed,Recognition of the important regulatory role of the immune system on the behavior of cancer cells has led to the awareness that manipulating the microenvironment could be used as therapeutic target. Indeed, stimulating the immune response against the cancer cells has been shown very successful in various cancer types , 13. HowWe investigated the intratumoral immune cell response, as well as the expression of these intracellular granzyme inhibitors in OPSCC as a potential mechanism for tumor cell immune evasion and correlated their expression with clinicopathological variables such as HPV status and overall survival.Our study cohort involved 288 patients with histologically proven primary OPSCC curatively treated at the University Medical Center Utrecht, the Netherlands, between 1997 and 2011 of which formalin fixed paraffin-embedded (FFPE) tissue blocks of the primary tumor were available in our pathology archives. The tissue blocks were incisional biopsies, as the treatment was mostly primary radiotherapy or chemoradiotherapy.www.federa.org). Anonymous or coded use of leftover tissue for scientific purposes is part of standard treatment agreement with patients in our center : 15.8–67.0). Of the total cohort of 274 patients, 151 (55 %) patients had died at the end of follow-up.Prognostic factors included in the univariate analysis were as follows: age, gender, tumor size (cT1-2 vs. cT3-4), smoking, alcohol use, nodal stage (cN0 vs. cN+), number of intratumor CD3+, CD8+, and CD4+ cells, SERPINB9 staining (positive vs. negative), SERPINB1 staining (high vs. low expression), and SERPINB4 staining (H-score high vs. low expression). In the total group, HPV-positivity, small tumor size (T1–T2), no lymph node metastasis, high number of CD3+ and CD8+ intratumor cells, and a low expression of SERPINB1 in tumor cells were all individually significant associated with an improved OS  are associated with an improved survival , 25. ConIn OPSCC, HPV status is a prognostic biomarker as HPV-positive tumors have a better long-term survival compared to HPV-negative OPSCC . MoreoveAs HPV-positive patients have an improved survival compared to those with HPV-negative OPSCC, it could be that less intensive treatment regimens in HPV-positive tumors could achieve similar efficacy with less side effects. Therefore, several clinical trials are currently investigating different ways to de-escalate treatment and still receive local control . HoweverSERPIN family members play pivotal roles in the inhibition of serine proteases in blood coagulation, mental diseases, and innate immune responses . StudiesIn selecting patients properly for immunotherapy, it is important to account for the presence of suppressive mechanisms of immune system. The granule exocytosis pathway is the major mechanism via which cytotoxic lymphocytes, including natural killer (NK) cells and cytotoxic T-lymphocytes, counteract virus-infected cells and tumor tissue. The principal mechanism of killing in this pathway is by the release of granules containing the pore-forming protein perforin and a family of structurally homologues serine proteases known as granzymes into the immunological synapse. Pore formation induced by perforin facilitates the introduction of granzymes into the cell. In the cell, they can induce apoptosis by the cleavage of intracellular substrates , 41. HumN = 10) in the HPV-positive group did not allow for multivariate analysis. Therefore, our results should be validated in a cohort with higher numbers of HPV-positive patients, and analyses should be stratified for therapy.The results of study should be viewed within the constraints of several limitations. First, TMA’s were used for the evaluation of the IHC stainings. Although TMA is accepted nowadays as a fast and accurate approach for the evaluation of IHC stainings in large groups, it must be denoted that it could introduce possible bias due to the high heterogeneity of HNSCC. Second, overexpression of SERPINB1 in HPV-positive OPSCC showed a significant association with worse survival, but the low number of patients and events (In conclusion, it is plausible that the immune system plays a more important role in carcinogenesis of virally induced oropharynx tumors. Consequently, HPV-positive patients with a high expression of SERPINB1 or low invasion of CD3+ cells should receive additional therapy instead of treatment de-escalation.