To achieve the ambitious 90–90-90 UNAIDS targets, access to routine viral load (VL) is critical. To measure VL, Rwanda has relied on a national reference laboratory for years. In 2014, a VL testing platform was implemented in a rural District in the Northern Province. Here we analyze the uptake of VL testing, identification of risks for detectable VL (≥1000 copies/ml), and the management of patients with a detectable VL.A retrospective cohort study of patients who started ART between July 2012 and June 2015 and followed until end December 2016. Using descriptive statistics, we describe the VL cascade, from VL uptake to the start of second-line ART in patients diagnosed with virological failure. We estimate predictors of having a detectable VL using logistic regression.n = 451) of patients tested, had a suppressed VL. Predictors of having a detectable VL included being male , being a sex worker , having a WHO clinical stage IV when starting ART , having had a previous detectable VL , and having had no VL before 2016 . Among patients with initial detectable VL between 2013 and 2016, 88% (n = 103) had a follow-up VL, of whom 60.2% (n = 62) suppressed their VL below 1000 copies/ml. The median time between the initial and follow-up VL was of 12.5 months (IQR: 8.7–19.0). Among patients with confirmed treatment failure, 63.4% (n = 26) started second-line ART within the study period.The uptake of VL testing increased progressively between 2013 and 2016, raising from 25.6% (39/152) in 2013 up to 93.2% (510/547) in 2016.In 2016, 88.5% (VL uptake increased after decentralizing VL testing in rural Rwanda. Virological suppression was high. An individualized follow up of patients at risk of non-suppression and a prompt management of patients with detectable VL may help to achieve and sustain the third global UNAIDS target: virological suppression in 90% of patients on ART. Despite significant efforts made in the prevention and the fight against HIV, the number of annual new HIV infections remains high . The subTo monitor progress towards achieving these targets, routine VL monitoring is indispensable. However, due to logistical and financial constraints, most sub-Saharan ART programs are unable to provide a regular access to VL testing for most of their patients on ART. A review showed important disparities between seven sub-Saharan Africa countries and revealed a number of countries without adequate capacities to perform VL test to all their patients on ART. The percentage of patients on ART with at least one VL test done ranged from 9% in Tanzania to 91% in South Africa .In the last 20 years, Rwanda has made tremendous progress in the fight against HIV, with a massive increase of HIV counseling and testing services and the decentralization of ART provision . Today, Since 2011, the Rwandan national guideline recommends routine annually VL testing in all patients on ART. However, its implementation has been challenged by the insufficient number of VL testing platforms, especially in the rural areas. In the last three years, the number of VL testing platforms has increased progressively. In addition, health care providers were trained on indications and the importance of having a routine VL . Two stuIn most resource-poor countries, VL testing is not decentralized. Hence, samples need to be transported to a central laboratory, which reduces accessibility to the VL testing . We therThis is a retrospective cohort study, using routinely collected HIV program data. Data were collected in one public hospital and one health center in the Northern Province of Rwanda.2, the country has the highest density of population in Africa. Still, 82% of the population lives in a rural area [Rwanda is a country with 11.5 million inhabitants, located in the Eastern African region. With 467 people per Kmn = 545) offering voluntary counselling and testing services (VCT), the provision of ART and prevention of mother to child transmission (PMTCT) services.In Rwanda, the HIV epidemic is stable, with a prevalence of 3% in the general population. The prevalence is higher in urban areas (6.3%) than in rural areas (2.4%), and in females (3.6%) than in males (2.2%) . The HIVRuhengeri hospital and Musanze health center are two neighboring health facilities, both located in the District of Musanze in the Northern Province of Rwanda. The HIV prevalence in this district is 2.7% .Ruhengeri district hospital is among the first health facilities that offered HIV services in Rwanda. HIV services are available since 2003. In 2016, the hospital counted 1605 patients actively followed up on ART. One medical doctor, four nurses and two social workers are assigned to provide HIV care. Since 2014, a VL testing platform using COBAS Amplipera and COBAS AmpliTaq machines has been available in Ruhengeri hospital. It serves to analyze samples from the hospital and neighboring health facilities. Using EDTA tubes, whole blood samples are sent from the health center to the hospital. A standardized external quality control is conducted twice a year in collaboration with the Center for Diseases Control in Atlanta-USA. The hospital uses OpenMRS software for the daily recording of patient’s data. Each patient has a unique TracNet identification number.Muhoza health center opened in May 2011. In 2016, the health center provided care for 973 active patients on ART. HIV care is provided by a team of three nurses and one social worker. Samples from the health center are sent to Ruhengeri district hospital for VL testing.All patients who were initiated on ART between the 1st of July 2012 and the 30th of June 2015 in both study sites were included in the study. Data were collected between the dates each patient started ART until the date of 31st of December 2016 which is the end of the study period. However, for patients who exited the program before the above mentioned date, we considered the date when those patients were transferred out, lost to follow-up, or reported as dead.Routine program data were collected from patient charts, ART registers and VL registers, available at both sites. This information was completed with data collected from the OpenMRS database. Collected data comprised socio-demographic and clinical variables.Socio-demographic variables included age, gender, area of residency, marital status, and current occupation. Clinical variables included the date of ART initiation, ART regimen, VL result, date of VL result, date of second-line ART initiation, treatment outcomes on 31/12/2016 .A patient is defined as ‘active on ART’ if he/she is in care at a given time, and has attended at least a pharmacy refill appointment within the last three-month period.The patient was ‘lost to follow-up’ (LTFU) if he/she didn’t attend the pharmacy refill appointment for more than three consecutive months.A patient was considered as ‘transferred out’, if for personal reasons, he decided to continue follow-up in a different health facility and received a transfer form from Ruhengeri hospital or Muhoza health center. Characteristics of patients transferred out were similar to those who remained in care.We defined a suppressed VL as each VL result less than 1000 copies/ml, and a detectable VL as a VL ≥ 1000 copies/ml. Virological failure was defined as two consecutive VL ≥ 1000 copies/ml, with at least a three-month interval between the initial and the control VL. Retention was calculated as the proportion of active patients among the study population at the end of the study period, excluding those who were transferred out from both the nominator and denominator (as their treatment outcomes could not be ascertained on 31st December 2016).The data collection was conducted by nurses working in the two study sites and data were encoded in an Excel database. A TracNet identification number was linked to each patient in the study, and the identity of patients was confidential at each step of the data collection.P < 0.05.Baseline characteristics of patients in our cohort were described using standard descriptive statistics. Univariate analysis was conducted using measures of frequency, median and interquartile range (IQR) for numeric variables and proportions for categorical variables. Multivariate analysis was conducted using logistic regression to establish a predictive model for having a detectable VL in 2016. The final model was retained when the likelihood ratio resulted in a In patients with a detectable initial VL between July 2012 and June 2015, we calculated the proportion that had a follow-up VL, and the delay between the high initial VL and the follow-up VL. According to the Rwanda national protocol, patients with a VL ≥ 1000 copies/ml should have a follow-up VL within six months. Patients have to be active in care to measure their VL. Therefore, we calculated the proportion of patients with a follow-up VL among those who were active six months after a high initial VL.Data were collected using Excel 2010 and analyzed using EPI info 3.5.4.No identifiers were encoded in the study database. This study was approved by the Ethics Committee of Ruhengeri provincial hospital as well as the Institutional Review Board of the Institute of Tropical Medicine of Antwerp, Belgium.A total of 775 patients enrolled on ART between July 2012 and June 2015 were included in the study. Most (67%) were female. The median age of patients enrolled on ART was 34 years (IQR: 27–41) (Table 3 (IQR: 265–531). At ART initiation, the vast majority of patients (88%) were prescribed TDF + 3TC + EFV combined regimen.Of 775 patients, about half were asymptomatic when they started ART (51.7%). The median CD4 count at ART initiation was 364 cells /mmn = 547) were active on ART; 21.4% (n = 166) were transferred out to another health facility, 2.7% (n = 21) died and 5.3% (n = 41) were lost to follow up. Retention on ART was 95.8% after one year on ART, 93.4 and 92% respectively after two and three years on ART. The median follow-up time for our cohort was 29 months (IQR: 21–41) on ART.At the end of the study period, 70.6% (p < 0.001), from 62.5% (195/312) to 70.2% (347/494) between 2014 and 2015 (p < 0.001), and from 70.2% (347/494) to 93.2% (510/547) between 2015 and 2016 (p < 0.0001). Yearly VL uptake increased significantly during the four consecutive years, between 2013 and 2016.The uptake of VL among patients on ART has been gradually increasing since 2013.  had a suppressed VL (< 1000 copies/ml) and 11.5% (n = 59) had a detectable VL (≥ 1000 copies/ml) , being a sex worker , having a WHO clinical stage IV when starting ART , having had a detectable VL before 2016 , and having had no VL before 2016   had an initial VL (first VL between July 2012 and June 2015), of which 146 (20.9%) patients had a high initial VL (≥ 1000 copies/ml). Out of 146 patients with a detectable VL, 29 exited the program without having a follow-up VL: 18 were transferred out, five died, and six were lost to follow-up. Among the 117 that were active 6 months after their high initial VL, 88.0% (n = 103) had a follow-up VL, of whom 60.2% (n = 62) had a VL below 1000 copies/ml  of patients with a high initial VL had a follow-up VL done. The median time between the initial and follow-up VL was 12.5 months. After a high initial VL, most (60.2%) patients had a suppressed follow-up VL. Most of patients (63.4%) with virologic failure (with a high follow-up VL) were initiated on second line ART.The uptake of VL in rural area of Rwanda was remarkably high when compared to other sub-Saharan Africa studies . StudiesVirological suppression in patients on ART (88.5%) was lower than what was reported in patients on ART in Botswana (95.6%) and Malawi (90.8%) but higher than in most other settings , 16, 17.In patients with detectable VL, the vast majority (88%) had a follow-up VL done. The median time between a detectable VL and a follow-up VL was 12.5 months IQR: 8.7–19.0). This period was twice as long as the period recommended by the national HIV guideline. Moreover, this contrasts with what was reported in South Africa, where a follow-up VL result was available after a median time of 4.9 months (IQR:2.8–6.1) . Session.0. This Among patients with a high initial VL, 60.2% suppressed the follow-up VL after adherence counseling and did not require any modification of their first-line ART regimen. Likely, enhanced adherence counselling helped patients to improve their adherence to treatment. This finding illustrates the benefit of a routine VL monitoring, as it allows to identify patients in need of a close follow up, and to monitor their adherence. Indeed, previous studies have shown that targeted adherence support can help patients to re-suppress their detectable VL and keep taking the first-line ART regimen , 22.In our study, almost two-third of our patients (63.4%) failing the first line ART were switched to the second-line ART before the end of the study period. This is quite similar to what was reported by a systematic review from 16 sub-Sahara African countries, which showed that 58% of patients with confirmed virologic failure switched to second-line ART . ReporteThe strengths of our study include that findings represent the real-life setting of an HIV program in rural Rwanda, as data were retrieved from routinely-collected patient-level program data. Moreover, we believe the findings are based on robust data. Data were retrieved from a prospectively updated electronic database, used for program monitoring. For the aim of this study, data were cleaned by comparing them with data from the individual patient files. No or very few values were lacking for most variables. Moreover, the vast majority of patients had effectively a VL done, which allowed us to evaluate the third UNAIDS target. However, there are some limitations as well. The two study sites, Ruhengeri hospital and Musanze health center are two neighboring health facilities, both located in the rural District of Musanze in the Northern Province of Rwanda. As there were no geographical barriers between the health facility where clinical care of patients was conducted and the site where VL testing was performed, the findings may not be generalizable to patients attending remote health facilities. Nevertheless, the findings show that when geographical barriers are minimized, routine VL monitoring is feasible to monitor the implementation of the ambitious UNAIDS 90–90-90 targets. Another limitation of the presented study is that adherence in terms of daily pill intake was not assessed. Moreover, the outcome of patients lost to follow-up was not known. As patients lost to follow up count generally among patients with low level of adherence on ART, they are more likely to have unsuppressed VL. It was also not possible to know the outcome of viral load testing for patients transferred out all along our study period.The uptake of routine VL monitoring was very high after decentralization of a VL testing platform in a rural area of Rwanda. The vast majority (88.5%) of patients tested had a suppressed VL. Among patients with detectable VL (≥1000 copies/ml), the majority of them re-suppressed after sessions of adherence counseling. Still, a close clinical follow-up of patients at risk of a detectable VL, such as patients with a previous detectable VL, patients who had a low CD4 count or advanced stage of disease when they started ART, female sex workers, may result in an earlier identification of virological failure. To shorten the time between a detectable and a follow-up VL, it is recommended an individualized patient monitoring and enhanced adherence counselling for patients with detectable VL. This may contribute to achieving and sustaining the third 90 UNAIDS target, aiming at having more than 90% of patients on ART with undetectable viral load.


Depression, prevalent among people living with HIV (PLWH) in Malawi, is associated with negative HIV patient outcomes and likely affects HIV medical management. Despite the high prevalence of depression, its management has not been integrated into HIV care in Malawi or most low-income countries.This study employs a pre-post design in two HIV clinics in Lilongwe, Malawi, to evaluate the effect of integrating depression management into routine HIV care on both mental health and HIV outcomes. Using a multiple baseline design, this study is examining mental health and HIV outcome data of adult (≥18 years) patients newly initiating ART who also have depression, comparing those entering care before and after the integration of depression screening and treatment into HIV care. The study is also collecting cost information to estimate the cost-effectiveness of the program in improving rates of depression remission and HIV treatment engagement and success.We anticipate that the study will generate evidence on the effect of depression management on HIV outcomes and the feasibility of integrating depression management into existing HIV care clinics. The results of the study will inform practice and policy decisions on integration of depression management in HIV care clinics in Malawi and related settings, and will help design a next-step strategy to scale-up integration to a larger scale.NCT03555669]. Retrospectively registered on 13 June 2018.ClinicalTrials.gov ID [ The Joint United Nations Programme on HIV/AIDS (UNAIDS) “90–90-90” goals provide a compelling roadmap toward achieving the end of the HIV epidemic. The UNAIDS plan, which has been embraced by the international public health community and much of sub-Saharan Africa, calls for 90% of those HIV-infected to be aware of their status, 90% of those aware of their status to be on sustained antiretroviral therapy (ART), and 90% of those on ART to be virally suppressed . AchieveComorbid depression renders people living with HIV vulnerable. Comorbid depression affects patients receiving ART  as it isMalawi, with a population of 17.5 million, has an adult HIV prevalence of 10%  and barrDepression is a prime target for strategies to improved HIV outcomes in Malawi. Depression treatment has been linked to improved ART adherence in a meta-analysis comparing those receiving vs. not receiving depression treatment , a conclThe purpose of this paper is to present the protocol for a quasi-experimental study designed to estimate the effect of the integration of depression treatment into routine HIV primary care on both mental health and HIV-related outcomes in Malawi.Our evaluation study has two objectives. The primary objective of this evaluation is to assess the impact of a pragmatic, scalable mental health treatment program on HIV care outcomes including retention in care and viral suppression. The secondary objective of this study is to evaluate the impact of the mental health treatment program on mental health outcomes, including depression remission and depression response.Specifically, our study is designed to evaluate the following hypotheses:H1 Compared to the period prior to the depression treatment program, adults with HIV and depression during the implementation of the treatment program will be more likely to be retained in HIV care and virally suppressed 6 months after ART initiation (primary outcome).H2 Compared to the period prior to the depression treatment program, adults with HIV and depression during the implementation of the treatment program will be more likely to be virally suppressed 6 months after ART initiation.H3 Compared to the period prior to the depression treatment program, adults with HIV and depression during the implementation of the treatment program will be more likely to be retained in HIV care 6 months after ART initiation.H4 Compared to the period prior to the depression treatment program, adults with HIV and depression during the implementation of the treatment program will have higher HIV care appointment adherence (proportion of scheduled visits that were attended) over the first 6 months on ART.H5 Compared to the period prior to the depression treatment program, adults with HIV and depression during the implementation of the treatment program will be more likely to have achieved depression remission 6 months after ART initiation.The study employs a multiple baseline evaluation design in two clinics to evaluate the impact of the integrated depression treatment program on HIV outcomes Fig. . The reaStudy activities will last 27 months. This period includes abstracting depression screening data on patients starting ART over a 20-month period and abstracting additional treatment and outcome data for an additional 7 months to identify follow-up viral load, appointment attendance, and depressive symptom outcomes. An additional 3 months is anticipated to complete data abstraction, analyses and dissemination activities.We are abstracting data on HIV and mental health outcomes for patients newly initiating ART who screen positive for depression Table .Table 1MDepressive severity is being measured using the Chichewa version  of the Patient Health Questionnaire (PHQ)-9. The score of 5–9 on the PHQ-9 is considered mild depression while that of 10 and above is moderate to severe depression . ProvideDepression Remission will be defined as a PHQ-9 score < 5 at 6 months.Retention in HIV care will be defined as being “on time” to all scheduled ART return visits in the first 6 months on treatment. “On time” will be defined as no more than 30 days late; a secondary, tighter definition will define “on time” as no more than 14 days late At ART initiation and each subsequent ART appointment, providers give patients a follow-up appointment date and a sufficient supply of ART. This information is recorded in the patient’s chart, called an HIV Mastercard, and in the Electronic Medical Record (EMR). However, generally for the first 6 months of care, new ART patients receive a 30-day supply of ART and a return appointment date in 30 days. Most often, patients need to attend monthly ART refill appointments for their first 6 months of care in order to maintain ART supply through 6 months.Consistent ART supply will be defined as never going more than 5 days without ART in the first 6 months, calculated from the days’ supply of ART dispensed at each appointment in the first 6 months and the time between appointments.Viral Suppression will be defined as a viral load < 1000 copies/mL at 6 months. A viral load threshold of 1000 copies/ml was chosen as this is the threshold used in the resource-limited Malawian health care system to guide decisions about treatment failure and second-line alternatives. Viral load testing is performed at Bwaila Hospital in Lilongwe using Abbott m2000 RealTime HIV-1 assay instruments with a lower limit of detection of 40 copies/mL. The results of the viral load test are recorded in the EMR.We integrated depression screening and treatment into ART care into two clinics in Lilongwe, Malawi using a multiple baseline design in two phases: a screening phase and a treatment phase.During the screening phase, HIV care providers screened patients for depression and monitored their depressive symptoms using the Patient Health Questionnaire-9 (PHQ-9). HIV care providers managed patients identified with depression according to existing care pathways, which included: 1) counselling by the HIV care provider; 2) referral to an on-site or off-site psychiatric nurse or other mental health specialist; or 3) in acute cases, transport to the outpatient psychiatric unit at the nearby district hospital.At the launch of the treatment phase, HIV care providers were trained in algorithm-guided antidepressant management ; additioWe adapted an ABCD antidepressant prescription model. ABCD is a resource-efficient, task-sharing model for delivering high quality, effective, safe antidepressant management in non-psychiatric settings. ABCD equips non-specialists  to safely and effectively prescribe and monitor antidepressants. The model trains staff to know what to measure , how and when to measure it, how to interpret the results, and what resulting course of action to pursue . Key features include (1) treatment decisions guided by regularly measured metrics of depressive severity, side effects, and adherence; (2) an algorithm summarizing best-practices care based on metrics; (3) a treatment goal of remission (full resolution of symptoms); (4) low starting dose followed by dose adjustments until remission, as long as the medication remains tolerable; (5) ensuring an adequate trial (six to eight weeks at moderate to high dose); and (6) regular structured supervision for continuous quality improvement.In our program, a patient initiates an antidepressant when identified with moderate to severe depressive severity (PHQ-9 score ≥ 10) combined with clinical confirmation of the presence of depression Fig. . Every fABCD has proven to be effective in primary care , 42 and We adapted a PST intervention developed for use in Zimbabwe called the Friendship Bench . PST is The original Friendship Bench consisted of task-shifting PST to lay health workers in Zimbabwe; requiring minimal training with structured supervision. The Friendship Bench PST intervention resulted in clinically meaningful improvement in mental health outcomes  and was In our program, the developers of the ‘Friendship Bench’ conducted a training for trainers who then went on to train a team of community health workers stationed at each clinic to deliver PST. The program built structures at both clinics for holding Friendship Bench PST sessions. Additionally, we staffed one Friendship Bench counsellor at each clinic throughout the duration of the program.Our intervention combined ABCD and PST into a stepped-care intervention with clinical response appropriate to the level of depressive severity Fig. . PatientWe are conducting the evaluation study at two public-sector primary care HIV clinics in Lilongwe, Malawi: Area 18 and Area 25 health clinics. These clinics are managed by Lilongwe District Health Office. More detailed description of the study setting has been published elsewhere.We are abstracting screening data for all consenting adult (≥18 years old) patients newly initiating ART and screened for depression at the time of ART initiation at the study sites during the study period. Additional depression treatment and outcome data are being abstracted for all patients who screen positive for mild, moderate, or severe depressive symptoms  , 57.We focused on two populations of interest. For the primary population of interest, all patients with depressive symptoms (PHQ-9 score ≥ 5), the original sample size was determined to provide 80% power to detect an 11 percentage point pre-post difference in the primary outcome. For the secondary  population of interest, all patients with moderate to severe depression (PHQ-9 score ≥ 10), the original sample size would provide the same power to detect an 18 percentage point pre-post difference. To achieve this power, we would require 645 patients with any depressive symptoms and 225 patients with moderate to severe depressive symptoms.n = 502) and the prevalence of moderate to severe depression was 6.3% (n = 131). This sample has 80% power to detect a slightly larger difference in the primary outcome of 12 percentage points (e.g. 60% vs. 72%) or a risk ratio of 1.2 for those with mild to severe depression. For those with moderate to severe depression, the sample has 80% power to detect an improvement in the primary outcome of 22 percentage points (e.g. 60% vs. 82%) or a risk ratio of 1.4.Based upon the historical rate of new ART patients (122 per month between the two clinics) and the expected prevalence of any depressive symptoms (35%) and moderate to severe depressive symptoms (12%) based on prior literature  7], we , we 7], Data on viral loads, HIV appointment attendance, and depressive symptoms is abstracted from electronic and paper medical records and the depression treatment registry at the sites. ART appointment data including the date of the appointment, the expected return date, and medication prescribed is recorded in the EMR, on a paper patient file , or both. The results of the viral load tests are recorded in the EMR. Data on patients’ depressive symptoms and depression treatment are only recorded on paper in an additional patient filed attached to the ART mastercard . The community health workers responsible for administering the Friendship Bench problem-solving therapy also maintain paper records  that capture engagement with the PST sessions. Cost information was collected using a time-audit approach to estimate costs of delivering the program, including the opportunity cost of training participation and the time providers and supervisors spend to deliver and supervise the program. Time-audit data was captured through a combination of direct observation of medical provider and counsellor encounters supplemented with self-reporting by counsellors.) This composite outcome was chosen as the primary outcome because it is the definition of ART clinical success in the Malawian ART treatment system; those not retained and those retained but not suppressed are both clinical failures. This main analysis will estimate the probability of retention and viral suppression 6 months after starting ART using a generalized linear model with binomial error distribution, adjusting for calendar time and potential confounders that are imbalanced between arms at baseline, potentially including age, sex, clinic, PHQ-9 score, and presence of suicidal ideation. We will model these associations using separate generalized linear models among two samples: (a) those with mild, moderate, or severe depression (PHQ-9 ≥ 5) and (b) those with moderate to severe depression (PHQ-9 ≥ 10).The primary program evaluation question is whether the depression treatment program results in improved HIV and mental health outcomes for patients with HIV and depression relative to the standard of care prior to program implementation. The main analysis will follow an “intent-to-treat” approach, classifying patients according to screening vs. treatment phase (unexposed vs. exposed to the program) without regard to actual treatment received. In our main analysis, we will define our primary outcome, retention in HIV care with viral suppression 6 months after starting ART, as never being more than 30 days late to any scheduled return visit in the first 6 months on ART and having a viral load < 1000 copies/mL at 6 months; patients not retained or not suppressed will be coded as a failure. Table  This comIn secondary analyses, we will consider ART care retention through 6 months (binary), number of ART appointments attended in the first 6 months (count), maintaining consistent ART supply through 6 months (binary), and viral suppression at 6 months among those retained (binary). Further, secondary analyses will take an “as treated” approach, comparing patients who receive treatment in the treatment phase to all patients during the screening phase; and an “as adequately treated” approach, comparing patients who receive adequate treatment (≥3 consecutive months of protocol-concordant antidepressant prescriptions or ≥ 4 counseling sessions) during the treatment phase to all patients during the screening phase. Data are not available on antidepressant adherence.  be adopted and integrated into routine care in public sector settings –61. We hThe program itself focuses on provider training to manage antidepressants and provide evidence-based counselling, and thus cannot be randomized at the individual level. Since patients do not see the same provider from one appointment to the next, in this context the intervention could not be randomized at the provider level either. A cluster-randomized trial was also beyond the resources available for this project. Given these constraints, a multiple baselines design provides stronger causal inference than would be available with a simple pre-post design. Multiple baseline designs have been advocated as one of the designs providing the strongest causal inference apart from the randomized trial .We anticipate that the study will generate evidence on the effect of depression management on HIV outcomes and the feasibility of integrating depression management into existing HIV care clinics. The results of the study will inform practice and policy decisions on integration of depression management in HIV care clinics in Malawi and similar low-income country settings, allowing design of a scale up strategy.


Various strategies to control the epidemic have been implemented, including the implementation of viral load (VL) testing to monitor HIV patients on ARVs. Like many resource limited settings, Kenya’s healthcare system faces serious challenges in effectively providing quality health services to its population. Increased investments to strengthen the country’s capacity to diagnose, monitor and treat diseases, particularly HIV and TB, continue to be made but are still inadequate in the face of global health goals like the UNAIDS 90:90:90 which require scaling up of VL tests amid existing constraints. In Kenya, there is an increase in the demand for VL tests amidst these existing constraints. The GeneXpert system is a diagnostic point-of-care technology that can quantify, amongst others, HIV VL. Currently, GeneXpert technology is widely distributed in Kenya for testing of tuberculosis. This study aimed to determine the economic and public health impact of incorporating VL test modules on the existing GeneXpert infrastructure. Markov models were constructed for different populations . The scenarios analysed were 100% centralized VL testing compared to 50% GeneXpert plus 50% centralized VL testing, with time horizons of 5 years for the adult and child populations, and 31 months for the pregnant population. Incremental effectiveness was measured in terms of the number of HIV transmissions or opportunistic infections avoided when implementing the GeneXpert scenario compared to a 100% centralized scenario. The model indicated that, for all three populations combined, the GeneXpert scenario resulted in 117 less HIV transmissions and 393 less opportunistic infections. The cost decreased by $21,978,755 for the non-pregnant and pregnant adults and $22,808,533 for non-pregnant adults, pregnant adults and children. The model showed that GeneXpert would cost less and be more effective in terms of total cost per HIV transmission avoided and the total cost per opportunistic infection avoided, except for the pregnant population, when considered separately.Kenya has the world’s 4 Kenya exhibits one of the worst epidemics of HIV and AIDS in the world , with apTo reduce HIV transmission rates, Kenya had outlined six goals in 2009 that were to be achieved by 2014. The objective of this strategy was to reduce the number of new HIV cases by using evidence-based approaches . There hPeople living with HIV (PLHIV) should follow the HIV care cascade to fully benefit from antiretroviral therapy (ART). This process begins with HIV testing and ends with monitoring the patients, ensuring that viral suppression has been achieved. PLHIV often do not adhere to the care cascade in the real-world setting . VariousA shortfall is observed in laboratory processing capacity in resource-limited settings. Consequently, many HIV treatment programs are designed in such a way that laboratory monitoring procedures are not prioritized . Focus iPatients on ART need regular monitoring to assess the effectiveness of treatment and identify emerging drug resistance which, when undetected, results in patients experiencing treatment failure with the consequence of needing more expensive second and third line regimens . This caRecent World Health Organization (WHO) guidelines suggest that viral load (VL) testing is the preferred way to monitor PLHIV who are treated with ART . ClinicaThe GeneXpert system is a point-of-care platform for rapid and simple-to-use nucleic acid amplification tests. The system is a modular, molecular diagnostic platform  that hasIn Africa, GeneXpert is deployed in many countries in both centralized and decentralized health care settings for the diagnosis of MTB and resistance to the TB drug, rifampicin . These cA GeneXpert test has been developed to quantify HIV VL using the same GeneXpert infrastructure.Given the existing GeneXpert infrastructure investment in Africa and the scalability of the technology (GeneXpert can perform any of the Cepheid molecular diagnostic tests on the same hardware), it is hypothesized that adding HIV VL tests to the existing GeneXpert infrastructure in Kenya could be a cost-effective way of implementing the WHO VL testing guidelines. This was also investigated to assist in effectively scaling up VL testing in line with the WHO 2016 guidelines. Models were constructed to investigate the economic and public health impact of adding VL testing to the existing GeneXpert infrastructure in Kenya.Markov models were constructed in Microsoft Excel with each model corresponding to one of three risk populations, namely non-pregnant adults (15 years and older), pregnant women and children (0 to 14 years). In this cohort model, two scenarios were compared for each of the three population risk pools. The model compared a scenario where 100% of VL testing is performed in centralized laboratories to a scenario where 50% of VL testing is done with GeneXpert plus 50% of VL testing is done in centralized laboratories.Six Markov models were constructed to incorporate each scenario for each population risk pool. Patients enter the model at 6 months on antiretroviral treatment (ARVs) (for the adult and child populations) or at 6 months gestation for the pregnant population. The time horizons for the models were 5 years for the adult and child populations, and 31 months for the pregnant population (corresponding to the last 3 months of pregnancy plus 28 months of breastfeeding thereafter). A cycle length of one month was used. Patients flow through the model with different probabilities, through different model states. At the time of developing the model, the Kenyan local currency to US dollar exchange rate was as follows: 1 Kenyan Shilling equals 0.00979435 US$ . This exThe child and adult populations of Kenya, split between males and females, are shown in The annual number of HIV-related deaths are shown in Loss to follow-up for centralized testing was calculated as the average between literature and programmatic data from the Kenya National AIDS and STI  Control Programme (NASCOP). Literature indicated that the percentage of patients who were 3 or more months late for a scheduled follow-up, but returned within 12 months, was 18.9%. The loss to follow-up rate for patients who were 12 or more months late for a scheduled follow-up, who did not return prior to database closure and were not known to have transferred out or died, was 20.2% [Based on these figures, 18.9% of patients had the opportunity to return to care at the next testing cycle. According to data for CD4 point-of-care (POC) testing vs centralized testing in Mozambique, loss to follow-up for POC testing was 48% less than for centralized testing . Using tWhen performing centralized tests, many tests are performed, but the results are not delivered to the patient, due to system errors. This percentage of test performed, where results are not received for centralized monitoring is 46% . This waThe risk of sexual transmission of HIV, based on the median VL and number of sexual contacts, was calculated based on Wilson et al. . An assuThe frequency of VL testing for virally suppressed and unsuppressed adults and children were every 12 months and every 3 months, respectively. The number of clinic visits per year for stable vs unstable patients are 2 and 6 visits respectively. These were based on Kenyan treatment guidelines .The run time for GeneXpert VL testing is 90 minutes. It was assumed that patients would therefore receive their results on the same day. The turnaround time for centralized VL testing is 15 days . It was The percentage of patients on ARVs with viral suppression  were 63.2% in the child population  and 84.2nd line treatment state. The transition matrix calculated the probability of receiving a retest, once a patient is 1st line confirmed, and with a VL above 1 000 copies/ml and multiplied this by the adherence figure for the patients who are not virally supressed. Thus, non-adherent patients will remain on 1st line therapy, and patients would only be switched to 2nd line if they were adherent, but still not virally suppressed.Based on the Kenya AIDS Indicator Survey (KAIS 2012)  the percThe number of cases of opportunistic infections (OI) per person-month was calculated by dividing the number of OI cases by the number of person-months . This waThe mother-to-child transmission rate at delivery was calculated for pregnant women with a VL <10,000 copies/ml (1.4%) and pregnant women with a VL ≥10,000 copies/ml (2.9%) . The motst and 2nd line ARVs for adults, pregnant women and children in Kenya are shown in Tables The costs of antiretroviral (ARV) unit costs for Kenya was sourced from the National HIV commodity plan for 2016 as the actual prices at which the Kenya Medical Supplies Agency (KEMSA) procures the medicine. For children, patients were assumed to be 3 to 15 years of age, with 50% of children with a weight <35 kg (average 20kg) and 50% of children with a weight ≥35 kg. It was assumed that the cost for efavirenz (EFV) 200 was 1/3 of the cost of EFV 600 as the cost for EFV 200 was not available. The daily cost of 1The cost of a centralized VL monitoring test ($25) was obtained from KOL opinion and based on internal data presented as a poster at IAS 2017 . The totThe costs of annual laboratory tests were obtained from the Kenya Fees Guidelines for Medical & Dental Practitioners  and convThe cost of a clinic visit was calculated as the average of clinical and nonclinical labour costs per outpatient visit at a cost of $4.55 .st line ARVs (for adults or children): $326.88 for adults and $257.41 for children. This is for one year of treatment for a newly diagnosed HIV-positive patient.The cost per transmission for patients who become HIV-positive was obtained from literature  , however, the number of patients who are awaiting tests are less, resulting in a decrease in OIs for 1st line patients. This resulted in increased costs for treating 1st and 2nd line ARV patients in the GeneXpert scenario, although monitoring cost is less in the GeneXpert scenario (due to the lower cost of the GeneXpert test). The GeneXpert scenario resulted in a total cost reduction of $829,777 , except when reducing the loss to follow-up for centralized testing and the viral suppression rate for adults by 5% (for HIV transmissions) or 10% (HIV transmission and opportunistic infections). From the sensitivity analyses it is evident that loss to follow-up for centralized testing and viral suppression rate are sensitive inputs to the model. Reducing the loss to follow-up for centralized testing by 5% and 10% resulted in an increase in the number of transmissions and opportunistic infections in the GeneXpert scenario compared to the 100% centralized scenario. The costs for the GeneXpert scenario, however, remained lower than the costs for the 100% centralized scenario.The percentage of test results not received for centralized monitoring is a startling 46%. Reducing this figure to 5% results in the GeneXpert scenario being more costly compared to 100% centralised scenario. The incremental cost per transmission and per OI is -R96 015 and -R32 282. This implies that improving the system to reduce the number of tests lost, the use of central laboratories can be cost-effective.Latest data on the NASCOP dashboard  indicateThe premise of this study was that, while VL monitoring is the preferred strategy for PLHIV, it is also the most expensive option. Given the sunk cost of capital investment of GeneXpert infrastructure in Africa, we therefore questioned whether the adoption of VL monitoring on the back of this existing infrastructure would be a cost-effective intervention strategy. We answered this question by developing an economic model that mimics the natural history of three risk pools of PLHIV and estimating the economic cost of the intervention on the one hand and the public health impact it achieves on the other hand. To our knowledge, this study is unique in that we could not find any evidence of similar studies that benefit from an existing decentralized POC laboratory infrastructure or that stratifies risk pools into three different cohorts. From a policy perspective, we furthermore believe that the results would be relevant to stakeholders who consider adopting the new WHO guidelines related to monitoring of PLHIV.st and 2nd line ART. Although more people were tested in these populations , less VL tests were ultimately performed , due to less tests being lost or not valid in the GeneXpert scenario.In the non-pregnant adult and child populations, introducing decentralized HIV VL testing decreased total and monitoring costs, transmissions and opportunistic infections. ARV treatment costs increased in these populations, pointing towards more patients remaining in care and being retained on 1In the pregnant population, 33,936 more women were tested, while 24,800 more VL tests were performed in the GeneXpert scenario. This might be due to the fact that more VL tests are required for pregnant adults (monthly VL monitoring tests) therefore, with increased non-pregnant adults in care, and more VL tests performed, both indicators increased. The benefit gained from POC technology and a reduction in loss to follow-up (LTF), is not that significant in the non-pregnant adults due to frequent testing in the centralized system as well.A modelling study performed on the Zimbabwean HIV-positive population found that avoidance of the proportion of failed tests or test results not received, differentiated care  and approaches that increase retention on ART impacts the most on cost-effectiveness of the model . These aIn another economic evaluation of viral load testing in rural Zimbabwe , three aFrom the cost-effectiveness analysis, the difference in turnaround time for NucliSENS EasyQ (BioMérieux) centralized VL testing vs GeneXpert VL testing was 9 days . In this scenario, GeneXpert VL testing is used specifically for the targeted 27% of ART patients who are in need of a fast turnaround time . Only interlaboratory turnaround time was considered. For GeneXpert VL testing, patients might have to visit clinics again as blood needs to be collected when the samples are sent to the laboratory due to the VL test being performed on plasma instead of dried blood spot (DBS) testing and the preparation and storage requirements for plasma. This was not considered. It is not clear how this would affect turnaround time or loss to follow-up.In a third Zimbabwean study, using the GeneXpert VL test resulted in shorter overall median turnaround time  . An erroAnother study used Activity-Based-Costing methods to compare point-of-care VL testing with centralized VL testing in terms of costs in Kenya . This stComparing results from this analysis against data reported in literature, showed that the annual number of new infections in adults was 71,034 [For children, the annual number of new infections is 6,613 from literature . In thisIn addition to the comparisons above, this model is not a dynamic model, therefore no additional patients are added to the model at any cycle, impacting incident cases at every cycle which could also contribute to the difference observed when compared against literature.For non-pregnant adults and children, introducing GeneXpert VL testing is a cost-effective way of implementing the WHO guidelines concerning monitoring of treatment failure and can be used to track progress toward the third 90-90-90 target.In the pregnant model, the number of VL tests performed per annum is every 6 months (twice a year) for suppressed patients and every month (12 times a year) for non-suppressed patients. For the adult and children populations, this input is every 12 months (once a year) for supressed patients or once every 3 months (4 times a year) for non-suppressed patients.In the pregnant population, due to the turnaround time for centralised testing, all VL tests performed has a 1-month delay until results are received. For non-supressed patients in a centralised model, patients will effectively only receive 6 VL tests per year, where it is determined whether a patient is LTF. In GeneXpert scenario, evaluations to determine LTF occurs every month . Thus, for the pregnant population, the GeneXpert model has more LTF patients (where there is a higher transmission probability), which implies more HIV transmission from those patients. If the number of VL tests performed in the pregnant model is set equal to that used in the adult and children population, less LTF patients are observed, and overall less transmissions for these patients. The same trends are observed for OIs and transmissions to babies, which, once VL testing frequency is changed, overall GeneXpert will show more benefit compared to centralised model.In terms of costs, the incremental direct cost  reduces significantly, although the incremental difference is smaller but still positive (more costly).The time horizon of the pregnant model is 31 months compared to 5 years. The 31 months represents pregnancy and breastfeeding. This cannot be changed as this is not a dynamic model, so once a baby is delivered and the mother is not breastfeeding anymore, she is not removed from the model. This might have an effect on overall costs and effectiveness observed in the pregnant model, compared to that observed in the non-pregnant adult and children populations.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Genvoya®  is a recent single regimen for the treatment of Human Immunodeficiency Virus (HIV). However, because of its complexity, it is difficult to predict drug interactions, especially when associated with HMG-CoA reductase inhibitors and/or in the setting of other comorbidities. We discuss the mechanisms of these potential drug interactions as the cause of rhabdomyolysis and acute kidney injury in the context of prior and current medication therapy with possible underlying liver and kidney dysfunction.We describe the case of a 54-year-old man diagnosed with HIV who developed severe rhabdomyolysis-induced anuric acute kidney injury (AKI) requiring renal replacement therapy following introduction of Genvoya® concomitantly with simvastatin, in the context of recently diagnosed hepatitis C and hepatitis A. Haemodialysis was continued over 5 weeks followed by progressive clinical and biological improvements. Five months later, a new antiretroviral regimen was started and has been well tolerated.https://www.hiv-druginteractions.org/checker), while for patients under hepatitis C-therapy, we advise to consult http://www.hep-druginteractions.org/. This case illustrates the importance of multidisciplinary collaboration in the treatment of HIV-positive patients because of their complexity, associated comorbidities and the potential of multiple drug-drug interactions potentially exacerbated by underlying liver and/or kidney dysfunction.Simvastatin, as well as lovastatin, because of their CYP3A4 metabolism, and to a lesser extent atorvastatin, which is only partially metabolized by CYP3A4, are the HMG-CoA reductase inhibitors with the greatest risk of drug interactions and should not be used in patients under HIV-therapy. Patients receiving HMG-CoA reductase inhibitors should be monitored regularly for the occurrence of muscular adverse effects and drug interactions should be considered with each new prescription or change in clinical status. There are many online tools that enable clinicians to rapidly check for drug interactions. We recommend the one from the University of Liverpool for patients under HIV-therapy ( The single treatment regimen Genvoya® associating elvitegravir, cobicistat, emtricitabine and tenofovir alafenamide (EVG/COBI/FTC/TAF) is a complete, simplified, efficient and safe combination pill for the treatment of Human Immunodeficiency Virus (HIV)-1 infection. It associates an integrase inhibitor (EVG), an integrase inhibitor booster (COBI) and two reverse transcriptase inhibitors (FTC and TAF). However, potential drug interactions may occur when taken together with 5-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors. Rhabdomyolysis-induced acute kidney injury (AKI) is a potential severe complication. Suttels and al. describe a case of rhabdomyolysis-induced acute kidney injury following use of similar HIV single treatment regimen (EVG/COBI/FTC/TDF) concomitantly with pravastatin/fenofibrate [A 54-year-old Caucasian man with known HIV infection for approximately 30 years had been treated with lamivudine, stavudine and indinavir since 1997. Under this therapy he was stable with an undetectable viral load. He also had dyslipidaemia which was treated with simvastatin 40 mg for many years. He presented to his primary care physician (PCP) with a 10-day history of asthenia, myalgia and jaundice. The initial laboratory revealed elevated liver enzymes (Alanine aminotransferase (ALAT) and Aspartate aminotransferase (ASAT) > 1000 U/l) and preserved kidney function (serum creatinine level of 79 μmol/l and estimated glomerular filtration rate (eGFR) of 90 ml/min/1.73 m2). He was diagnosed with acute hepatitis A and active hepatitis C genotype 1a, which was a new diagnosis. A second visit to his PCP 6 days later showed improvement of the liver enzymes. As stavudine had been withdrawn from the Swiss market, his HIV therapy was switched to Genvoya® at that time. The patient had declined referral to an HIV-specialist for confidentiality reasons.Ten days after this medication switch, the patient presented to the emergency department (ED) with worsening myalgia and asthenia and could barely walk. He reported a reduced urine output during the prior days. There was no history of trauma, prolonged immobilization, convulsions or consumption of alcohol or illicit substances. On evaluation in the ED, the patient was slightly hypertensive at 144/84 mmHg. He presented with mucocutaneous jaundice. The cardiopulmonary status was unremarkable and the patient had no edema of the extremities. Abdominal examination revealed hepatomegaly 2 cm below the costal margin. Muscle strength was severely diminished, mostly in the axial muscles.Initial laboratory evaluation on admission revealed elevated creatinine kinase (CK): 185190 U/I, creatinine: 553 μmol/l, phosphate: 3.03 mmol/l, potassium: 7.2 mmol/l, ASAT: 7017 U/I, ALAT: 2881 U/I, gamma-glutamyl transferase (GGT) 198 U/I, and total bilirubin: 130 μmol/l  fluids and medical treatment for life-threatening hyperkalaemia with electrocardiographic changes. Because of refractory hyperkalaemia and metabolic acidosis, he was initiated on haemodialysis in a tertiary care setting. His antiretroviral and lipid lowering therapies were discontinued.Intermittent haemodialysis was continued for over 5 weeks followed by progressive improvement of renal function and diuresis, serum electrolytes, liver enzymes and CK. His viral load remained low. Five months later, his creatinine had normalised to 77 μmol/l and antiretroviral therapy was restarted by an HIV-specialist with emtricitabine, tenofovir alafenamide, etravirine, ritonavir and duranavir. These were well tolerated both clinically and metabolically after several weeks. His lipid panel at the time did not warrant further treatment with a statin and he has yet to be started on treatment for hepatitis C.The patient described here presented with acute kidney injury AKIN stage III induced by severe rhabdomyolysis that was diagnosed 10 days after the introduction of Genvoya®. The main causes of rhabdomyolysis comprise crush injury, burns, infections, medications, drug use, and extreme exercise . Acute kThe Naranjo Adverse Drug Reaction Probability Score , which aStatins (HMG-CoA reductase inhibitors) act as direct myotoxins , althougSimvastatin, the statin prescribed for our patient, is one of the three metabolized by cytochrome P450 3A4 (CYP3A4). Its potential for interaction with potent inhibitors of CYP3A, such as COBI, is one of the highest. COBI, one of the four constituents of Genvoya®, lacks intrinsic anti-HIV activity. It is used as a booster of EVG, allowing for higher plasma concentrations of the latter with lower administered doses, thereby reducing secondary effects   or http://www.hep-druginteractions.org/ for patients under hepatitis C-therapy which are easily available online should become routine in the clinic.Medication interactions are avoidable. AKI is a severe complication and even after recovery is associated with increased long term mortality and risk of chronic kidney disease . This caIt is important to recall that drug interactions associated with CYP3A4 metabolism occur with a wide range of medications including commonly used drugs such as, among many others, amiodarone, calcium-channel blockers, azole antifungals and macrolides antibiotics (Table This case reveals the complexity of the treatment of HIV-positive patients who are at risk of multiple potential drug interactions, especially when statins are associated with fixed anti-retroviral drug combinations and in the setting of potential liver and/or kidney dysfunction. Simvastatin, lovastatin and, to a lesser extent atorvastatin, are the HMG-CoA reductase inhibitors with the greatest risk of drug interactions . They sh


Safety was assessed by rates of LPV/r-related adverse events (AEs), including lipid disorder, severe abnormal liver function, myelosuppression, and renal function. Between 2009 and 2016, 1196 participants  were ultimately enrolled. All patients had been on LPV/r-based second-line ART treatment for more than one year after failure of any first-line ART regimen. Overall CD4+T cell counts increased from 138 cells/mm3 to 475 cells/mm3 and 37.2% of all participants reached CD4 recovery. Viral suppression rates dramatically increased at the end of the first year  and gradually increased during follow-up . The most frequently reported AEs were LPV/r-induced lipid disorders with no obvious increase on LDL-C at follow-up visits. This is the first real-world LPV/r-based second-line treatment study to cover such a large population in China. These results provide strong clinical evidence that LPV/r-based second-line ART is effective in increasing CD4+T cell counts and viral suppression rates with tolerable side effects in HIV-infected adults in China in whom first-line treatment had failed.The World Health Organization guidelines recommend lopinavir/ritonavir (LPV/r) as a second-line antiretroviral therapy (ART) for HIV-infected adults in middle-income and low-income countries as a protease inhibitor boost based on clinical trials; however, the real-world safety and efficacy remain unknown. Therefore, we conducted a large-scale, multicenter retrospective cohort study to evaluate the efficacy and safety of LPV/r-based ART among HIV-infected adults in China in whom first-line therapy failed. The data were obtained from a national database covering 17 clinics in China for six years of follow-up from 2009 to 2016. Failure of first-line treatment was determined according to a viral load at least 400 copies/ml at week 48, non-completers at week 48 for any reason, and those who switched ART before week 48 for any reason such as side effects. Treatment effectiveness was assessed by the rate of CD4 Over two thirds of new cases of HIV diagnosed globally in 2017 were estimated to have occurred in resource-limited areas, including eastern and southern Africa, western and central Africa, and Latin America . ThousanHowever, an increasing number of HIV-infected adults have shown ﬁrst-line regimen failure, requiring a switch to second-line therapy . MoreoveThe WHO guidelines recommend second-line combination ART with a ritonavir-boosted protease inhibitor  combined with at least two nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs). Lopinavir/ritonavir (LPV/r) is currently widely used in middle-income and low-income countries, such as China and South Africa, based on demonstrated effectiveness and safety with respect to immunological restoration and tolerable side-effects in ART-naïve and experienced patients in combination with other ART drugs in clinical trials . CurrentMany factors can potentially influence real-world efficacy , includiTherefore, we conducted the present Chinese multi-center real-world cohort study to provide suitable data on the efficacy and safety of second-line ART with LPV/r for all patients in whom ﬁrst-line ART had failed, which can help develop standard guidelines for treatment.+T cell counts, viral load, and WHO stage), ART treatment history, and HIV-related diseases. There was no adverse events (AEs) information in the national databases; therefore, details on AEs were collected separately according to participants’ medical records.This large-scale multi-center retrospective study was conducted using data collected from a national database from 2009 to 2016 across 17 clinics in China . This stThe efficacy of the second-line LPV/r regimen was evaluated according to the immunological and virological responses at baseline and at 6, 12, and 72 months on ART.+T cell count more than 500 cells/mm3, and the percentage of viral suppression was defined as a viral load below 50 or 400 copies/ml. WHO-defined stage IV disease was determined according to the WHO clinical staging of HIV disease in adults. Factors related to recovery of CD4+T cell counts and WHO-defined stage IV disease were also analyzed. The safety of second-line LPV/r regimens was evaluated according to the rates of drug-related AEs  for good immune recovery were estimated with 95% confidence intervals (CIs). All P values were two-sided, and P < 0.05 was considered statistically significant. The data were analyzed using SPSS version 24.0 for Windows .Baseline demographic and clinical data were stratified according to the baseline age of participants (<50 years or ≥50 years). Descriptive statistics are presented as medians with interquartile ranges (IQRs) or counts with proportions as appropriate. Two-sample Between 2009 and 2016, 4006 patients used LPV/r as a second-line drug. Among these, 2078 patients with a viral load below 400 copies/ml at baseline were excluded from the study, leaving a total of 1928 patients in the first-line treatment failure group. A further 53 patients below 18 years of age, 556 patients who were undergoing the second-line treatment for less than 1 year, and 23 duplicate records were excluded, resulting in a total 1196 participants who failed any first-line ART regimen and switched to LPV/r-based second-line ART enrolled in the study.P < 0.001). Detailed information of the other differences in baseline characteristics according to age group is shown in The median age of the eligible participants was 36 years . There were fewer co-infections with hepatitis C virus among those older than 50 years than among the younger patients. Distributions of routes of transmission also significantly differed among the two age groups -containing treatment. In the final multivariate model, factors associated with CD4 recovery included baseline CD4+T cell count and age  and showed a slow increase during following-up  in the first year after switching due to failure of first-line treatment, and remained stable during follow-up visits in this real-world setting, which is consistent with the results of previous observational studies and RCTs , Major Project of Beijing Municipal Science and Technology Committee , the National Natural Science Foundation of China (No. 81571973), and Beijing Key Laboratory (No.BZ0089).This work was supported by the Chinese Government 13The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HIV drug resistance (HIVDR) poses a threat to the HIV epidemic control in Zambia especially in sub-populations such as the 15–24 years where there is poor virological suppression. Understanding the prevalence and patterns of HIVDR in this population (15–24 years) will contribute to defining effective antiretroviral therapy (ART) regimens, improving clinical decision making, and supporting behavioral change interventions needed to achieve HIV epidemic control.A cross-sectional analysis of study enrollment data from the Project YES! Youth Engaging for Success randomized controlled trial was conducted. Participants were 15 to 24 years old, who knew their HIV status, and had been on ART for at least 6 months. All participants completed a survey and underwent viral load (VL) testing. Participants with viral failure  underwent HIVDR testing which included analysis of mutations in the protease and reverse transcriptase genes.A total of 99 out of 273 analyzed participants receiving ART had VL failure, of whom 77 had successful HIVDR amplification and analysis. Out of the 77, 75% (58) had at least one drug resistant mutation, among which 83% (48/58) required a drug change. Among the 58 with HIVDR mutations, the prevalence of at least one HIVDR mutation to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) were 81%, 65.5% and 1.7%. The mutation M184V which confers resistance to NRTI drugs of lamivudine (3TC) and emtricitabine (FTC) was the most common (81%) among NRTI associated mutations followed by K65R (34.5%) which is associated with both tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide fumarate (TAF) resistance. Thymidine analogue mutations (TAMs) which confer resistance primarily to zidovudine (AZT), stavudine (d4T) and other NRTIs were observed at 32.8%. Common TAMs were K70RTQNE (32.8%), K219QE (22.4%), D67N (17.2%) and T215IT (15.5%). The most common NNRTI associated mutation was the K103N (65.5%) which confers resistance to both efavirenz (EFV) and nevirapine (NVP). There was a relatively high occurrence of other NNRTI mutations V106A (36.2%), as well as Y188C (36.2%) and Y181C (36.2%) which confer resistance to etravirine.There is a high prevalence of HIVDR including TAMs despite majority of these patients (90.48%) being on AZT or d4T sparing first line ART among the youth. Emergence of these mutations including the NNRTI associated mutations (Y181C and Y188C) may compromise future second- and third-line regimens in the absence of routine HIVDR testing. HIVDR monitoring at start of ART or at first-line failure can better inform clinical decision making and ART programing. Zambian adults prior to widespread use of ART in Zambia [Zambian adults failing first line ART had HIVDR in 2009–2012 [The introduction of universal access to antiretroviral therapy (ART) for chronic HIV care management has been one of the greatest achievements of the last two decades for sub-Saharan Africa (SSA) where the burden of HIV is highest . In Zambn Zambia . Subsequn Zambia . Another009–2012 . AnotherWhile HIVDR is recognized as a serious threat to attaining the 90-90-90 goals , 4, therThis paucity of adolescent data is a gap in our knowledge. Pediatric HIV treatment has led to an increased survival of perinatally infected children and adolescents creating a new generation of heavily treatment experienced adolescents and young adults , 12, 13.The 2019 Zambia Population-based HIV Impact Assessment (ZAMPHIA) report highlights these risks with youth achieving the lowest viral suppression rates, 34% in females and 35.7% in males aged 15–24 years compared to 73.0% and 74% in male and female adults 45–59 years . PrelimiWe thus analyzed baseline study enrollment data collected through a randomized controlled trial entitled “Project YES! Youth Engaging for Success” in order to determine the prevalence and characteristics of HIV drug resistance among ART- experienced youth, aged 15 to 24 years, attending four HIV care clinics in Ndola, Zambia.The cross-sectional analysis was conducted using study enrollment data from Project YES! a randomized controlled trial that tested a clinic-based peer mentoring program for HIV-positive adolescents and young adults (AYA) in four clinics in Ndola, Zambia. Project YES! study methods are described in detail elsewhere  . In brieThe recommended first-line ART regimens in Zambia at the time of this study comprised of two nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs) along with a non-nucleoside reverse transcriptase inhibitor (NNRTI). The two NRTIs/NtRTIs were tenofovir disoproxil fumarate (TDF) or zidovudine (ZDV) or abacavir (ABC) plus either lamivudine (3TC) or emtricitabine (FTC) whereas the NNRTI was either efavirenz (EFV) or nevirapine (NVP). The second line regimens comprised of two NRTIs (TDF or ABC or ZDV plus 3TC or FTC) sequentially selected based on which NRTIs/NtRTIs were used in first line and a boosted protease inhibitor (PI), lopinavir-ritonavir (LPV-r) or atazanavir-ritonavir (ATV-r). Third line ART typically consisted of tailor-made regimen based on HIVDR results. The typical drugs that were available in public health facilities offering third line ART at the time of the study included the NNRTI etravarine (rilpivarine and doravarine were not yet available) and the NRTIs lamivudine and emtricitabine (for M184V mutations). PIs included lopinavir and atazanavir for patients who demonstrated sensitivity on HIVDR testing. However, in some cases the PI darunavir boosted by ritonavir was available as a third line option. Finally, raltegravir was the only integrase inhibitor available for use as a third line ART. Dolutagravir had not been introduced to the public health sector until late in the intervention phase of this study and the HIVDR testing did not test for integrase inhibitor gene regions.There had been several ART policy changes that affected ART regimens before the study. These had been implemented as part of Ministry of Health directives to health facilities over time and included drug switches from stavudine to zidovudine, nevirapine to efavirenz and more recently tenofovir to abacavir based regimes for children and adolescents. Occasionally, the drug changes were driven by drug availability, but this was rather rare. Unfortunately, there has been inadequate documentation of reasons for drug changes in most study participants medical charts.http://hivdb.stanford.edu/. HIVDR was defined as the presence of at least one drug resistance–associated mutation (DRM) according to the World Health Organizations (WHO) 2009 Surveillance Drug Resistance Mutation list, using the Stanford Calibrated Population Resistance analysis tool . A cross-sectional analysis of all HIVDR results at study enrollment was done to determine HIVDR prevalence and characteristics among adolescents and young adults with virologic treatment failure.Blood samples for HIV viral load testing were obtained and collected in EDTA anticoagulated bottles. HIV viral load was determined using the Cobas Ampliprep/CobasTaqman 96 machine . Blood samples with a viral load of >1000 copies/mL (virologic treatment failure) were further subjected to an HIV drug resistance test using an Applied Biosystems Genetic Analyzer model 3500XL . HIV-1 RNA was extracted from 140 milliliter of plasma using the Qiagene QiAmp viral RNA mini kit . Genotyping encompassed protease and codons 1–230 of reverse-transcriptase genes. Sequences were assembled and manually edited using Sequencer version 4.5 software . The outcomes of the HIV resistance tests were reported using the Stanford HIV drug resistance database at Ethics approval was obtained from the ERES Converge Research Ethics Committee and the Johns Hopkins University Institutional Review Board (JHU-IRB) for the parent Project YES! study. The Ministry of Health -Zambia, through the National Health Research Authority, approved the conduct of the study in the four sites.From December 2017 through May 2018, a total of 276 youth age 15 and 24 years from four clinics were recruited into the parent Project YES! study. One participant had missing study enrollment viral load results and two participants were removed from the analysis as data cleaning revealed they had been on ART for less than 6 months at start of the study. Among the 273 participants for whom study enrollment HIV-1 RNA viral load results were available, 99 participants (36.4%) had viral load ≥1000 copies/mL, signifying treatment failure. Out of these 99 participants, HIVDR amplification was successful in 77.8% (n = 77). A number of specimens with borderline viral loads failed to amplify. Resistance results were originally entered into a Microsoft® Excel 2010  database and imported into SAS 9.4  for further data cleaning, recoding and statistical analyses. The data included both continuous and categorical variables. Continuous variables, such as age, were described using means and standard deviations, and medians with value ranges and interquartile ranges (IQR’s). Categorical variables, such as presence of a particular mutation, were described using frequency and percentage distributions.Among the 77 youth with an HIVDR test result, 51.7% were female, 48.3% were aged 3–10 years and 52% were aged 11–24 years at the start of ART, the mean age at the time of the study was 18.4 years, and 72% self-reported to have been perinatally infected . Based oHIV Drug Resistance associated Mutations (DRM) were identified in 75.3% (58/77) of all successful sequences. Of the 77 youth with HIVDR results, 64 were on first line and 13 were on second line ART . The preAmong the 58 patients with any resistance 48 (83%) had resistance to a drug they were currently taking and needed to switch drugs to an effective ART regimen . Out of Of the 45 who needed to switch to second line, 42 where on tenofovir based regimen, 2 on abacavir and 1 on zidovudine based regimens. Of these (n = 45), 19 had the K65R mutation that confers resistance to tenofovir and abacavir but potentiates the effectiveness of zidovudine as a second line option. Additionally, 13.3% (6 out of the 45) needing first line drug change had the type 2 TAMs combination of T215Y, D67N and K70RTQNE which in combination diminish the effectiveness of zidovudine.Drug mutations were categorized based on the WHO2017 drug mutation list and the Stanford HIV drug resistance database . The mosOverall, the 10 most common mutations were M184V (81%), K103N (65.5%), Y188C (36.2%), Y181C (36.2%), V106A (36.2), K65R (34.5%), K70RTQNE (32.8%), G190ASV (31.0%), K101EHP (31.0%) and E138AGQ (29.3%). The prevalence of mutations Y188C and Y181C was at 36.2% for and these  confer resistance to the NNRTI drug etravirine. Among the NRTI mutations, the most common Thymidine Analogue Mutations (TAM’s) were K70RTQNE (32.8%), K219QE (22.4%), D67N (17.2%), T215IT (15.5%) and M41L (5.2%). The ten most common mutations to the drug class NNRTI included K103N (65.5%), V106A (36.2%), Y188C/L (36.2%), Y181C/V(36.2%), G190ASV(31%), K101EHP(31%), E138AGQ(29.3%), A98G(22.4%), P225H(20.7%), and V108I(17.2%). The N88S was the only major PI mutation (1.7%) and there were three PI-Accessory mutations of L10LF (1.7%), K20T (1.7%) and Q58E (5.2%). Additional information of mutations by ART regimen is contained in Figs Our study provides critical data on resistance among adolescents and young adults with viral failure and attending clinical care in Ndola, Zambia and it has three key findings. The study population had similar characteristics for study participants who had successful HIVDR and those who did not. First, we observed a high level of HIV drug resistance, with three quarters (75.3%) of participants with viral load failure having at least one HIVDR mutation, and more than half (62%) showing drug resistance to at least one of their current ARV drugs. This suggests that almost two-thirds of youth with VL failure need an ART drug change while over a third may be failing due to reasons other than HIVDR, such as incomplete adherence and sub therapeutic dosing. It is also possible that participants with incomplete adherence could have HIVDR but with reversion to wild type. The phenomenon of reversion HIV to wild type in the absence of sufficient drug pressure may complicate the interpretation of HIVDR results for study participants who had virologic failure but no HIVDR. Reversion to HIV wild type has been documented in various settings and has been acknowledged as a challenge to interpretation of HIVDR results , 25.High levels of HIVDR mutations are not unique to AYA or Zambia. Several studies among adults from Zambia, South Africa, Togo and Tanzania, have shown a range of HIVDR from 84–98% , 27. TheFurther, our study showed that most participants who had HIVDR to a current regimen were failing first-line ART  and needed a drug change to a second line regimen. This data highlights the potential role of delayed clinical decision making in this population. Interpretation of patient laboratory results solely lies with the attending health care workers. Factors that may contribute to delayed decision making may include inaccessibility of routine viral load test, delays in the turn-around times for viral load results , as well as un-timely interpretation of results that are in patient medical records. There is therefore urgent need to strengthen mechanisms to ensure timely availability of viral load results, improve tracking mechanisms for results, and clinical supervision of health care workers to support timely decision making.Secondly, this study found the prevalence of HIVDR mutations to first line NNRTI and NRTI was common (81% and 65% respectively). Only one patient (1.7%) had major protease inhibitor (PI) resistance mutation. This high prevalence of HIVDR mutations to NNRTI supports the 2020 Zambian HIV guidelines on the use of drugs with high genetic barriers, such as dolutegravir (DTG) for all age groups and protease inhibitors (lopinavir-ritonavir) for specific populations such as children ineligible for DTG  for firsAs our study sampled ART treatment experienced AYA, we do not know the prevalence of primary transmitted resistance. However, studies from Zambia and sub-Saharan region have consistently shown an increasing prevalence of primary NNRTI resistance among adults (18 years or more) and pregnant women , 16, 29.In terms of HIVDR to NRTI, the prevalence of any TAMs was at 32.8% and the most common TAM’s were K70R/T/Q/N/E (32.8%), K219Q/E (22.4%), D67N (17.2%), T215IT (15.5%) and M41L (5.2%). TAM’s confer resistance to zidovudine, stavudine and other NRTI. When compared to earlier studies from Zambia [It is concerning to see the high prevalence of TAMs despite the majority of these patients 90.4%) having been on zidovudine or stavudine sparing first line ART at the time of recruitment into the study. Since these patients were on tenofovir or abacavir, there is possibility that the TAMs observed were due to transmitted resistance or to archived mutations from prior drug switches/exposures. Transmitted resistance and archived mutations have reported as possible causes of HIVDR , 30. The0.4% haviThe third major finding of this study was the high prevalence of the Y188CE (36.2%) and Y181C (36.2%) mutations, indicating resistance to third line therapy etravirine and rilpivirine, to which none of our study participants had ever been exposed to. As a result, even before the restricted availability of etravirine in Zambia, more than one out of three (36%) patients with HIVDR may potentially be resistant to the only salvage NNRTI available in the public health sector in Zambia. The high prevalence of the Y181C and Y188C mutations has been demonstrated in earlier studies from the region including Zambia , 26, 11.This analysis was a cross-sectional analysis among treatment-experienced participants and could not determine primary HIVDR or causal factors leading to HIVDR among AYA living with HIV. Data was also not collected on the PMTCT exposure/prior nevirapine use among participants or their parents. Non the less, we believe that our data makes a significant contribution to the database on prevalence and characteristics of HIVDR among AYA which remains limited in literature.HIVDR represents a serious challenge for Zambia’s national and regional health care systems. Many AYA with viral load failure may be resistant to their current regimens and in need of regimen change. Closer monitoring of drug resistant mutations at the start of HIV treatment or at first line failure can better inform clinical decision making and HIV treatment regimen programing.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


We aimed to investigate the role of plasma ADA as a biomarker of inflammation in treatment-naïve HIV patients who received tenofovir or another nucleoside analog for comparison. Ninety-two treatment-naïve patients were included in the study and grouped by treatment, i.e., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF) or Triumeq. ADA activity was measured in plasma and cytokines were analyzed by MILLIPLEX® MAP-Luminex® Technology. Plasma concentration of monocytes and neutrophils was measured at 0, 3, and 12 months post-treatment. Treatment-naïve HIV patients had increased ADA concentrations (over 15 U/L) that decreased after treatment with TAF and Triumeq, though this did not occur in TDF-treated patients. However, all groups exhibited a pro-inflammatory systemic profile at 12 months of treatment. Plasma GM-CSF levels decreased after 12 months of treatment in the TDF group, with a concomitant decrease in blood monocyte count, and a negative correlation with ADA values was found. In conclusion, ADA levels may be modulated by antiretroviral therapy in HIV patients, possibly affecting inflammatory status.Plasma levels of adenosine deaminase (ADA), an enzyme that deaminates adenosine to inosine, are increased during inflammation. An increase in ADA activity occurs with lower human immunodeficiency virus (HIV) viral load and higher CD4 Complex interactions between immune cells and soluble factors are responsible for the inflammatory response triggered to protect against microorganisms or at the site of injury. One such soluble factor is adenosine, a purine nucleoside that, when accumulated in the extracellular space, modulates the immune response and prevents inflammatory tissue damage . The immADA regulates extracellular adenosine levels and consequently controls adenosine receptor stimulation and balances the immunosuppressing effects of adenosine, thus making it an indicator of cellular immunity. ADA is essential for proliferation and differentiation of lymphoid cells, especially T cells, and aids in the maturation of monocytes into macrophages . AdenosiIL1β, TNFα, MIP1α/CCL3, and IL10 mRNA expression [The main cellular targets of HIV (human immunodeficiency virus) are CD4+ cells, especially the CD4 subset of T cells, peripheral blood monocytes, and tissue resident macrophages . Defectipression  and exprpression .+ T cell counts are strongly correlated with an increase in ADA [In HIV, the viral glycoprotein gp120 prevents interaction between ADA and the costimulatory signal for TCR-mediated T cell activation (CD26) ,24. Lowee in ADA .CD39 gene polymorphism modify the progression to AIDS, thus implicating adenosine production in disease progression [HIV patients have a higher frequency of Treg cells expressing CD39 (an ectonucleotidase that converts ATP and ADP into AMP). In these patients, effector T cells show higher in vitro sensitivity to the suppressive effect of adenosine due to higher expression of the adenosine A2A receptor . Moreovegression . Ex vivogression .In the present study, we determine whether plasma levels of ADA can be used as a marker of the disease. Furthermore, we study cytokine levels and their relationship to inflammatory response after 12 months of treatment with tenofovir compositions TDF and TAF compared to another nucleoside antiretroviral drug  in naïve HIV patients.p < 0.05) (p = ns), at which time 50% of patients were over the limit of 15 U/L  U/L) (p = ns. vs baseline); Triumeq remained stable  U/L) (p = ns vs. baseline), with only 20% of patients over the limit (p < 0.0001)  , Table 1he limit , Table 1 0.0001) . TDF cau 0.0001) , althougTwelve months after antiretroviral treatment initiation, we observed a predominant pro-inflammatory profile among patients treated with either form of tenofovir (TDF and TAF) and, to a lesser extent, with Triumeq.p = ns); meanwhile, INFγ decreased 11-fold in patients treated with Triumeq (p = ns) when compared to baseline levels   . No sign= 0.036) . Comparip < 0.0001), together with a tendency to decrease in IL5 . On the other hand, IL7 increased 56.2-fold when compared to baseline (p > 0.05) (p = 0.055) when compared to TAF (p < 0.05) and GM-CSF showed a tendency to increase . For its part, IL7 expression decreased 26.4 from baseline (p = ns)  . This ded to TAF . Surpris(p = ns) . These cρ = 0.75 p = 0.012 and ρ = 0.59 p = 0.049)  .p < 0.05 and p = ns, respectively). Interestingly, in the case of TDF, the number of cells remained fairly stable   A.When we evaluated neutrophil concentration in plasma B, we obsρ = –0.70 p = 0.008)  C.In this prospective study, we studied, on the one hand, cytokine levels as inflammatory response variables after 12 months of treatment with tenofovir compositions (TDF and TAF) in previously naïve HIV patients compared to another nucleoside antiretroviral drug (Triumeq), and on the other, we studied plasma levels of ADA to investigate whether this value can be used as a marker of the disease. We observed an increase in ADA values  during the 12 months of treatment in the presence of TDF that did not occur with TAF or Triumeq. We also found a proinflammatory profile in TDF- and TAF-treated HIV patients, with a decrease in GM-CSF for TDF group.Unlike previous studies, our work reports differences in ADA values and cytokine expression in naïve HIV-infected individuals, focusing on early treatment time points (3 and 12 months). Previous research has assessed circulating inflammatory mediators based on their reported associations with systemic inflammation .In physiologic conditions, adenosine levels in the extracellular space are low, and they increase under tissue damage, inflammation, infection, hypoxia, and stressors . AdenosiIn HIV, purine metabolism is involved in the onset of infection, the maintenance of inflammation, and in immune activation. HIV infection induces changes in immune cells, in adenosine concentrations, and this induces the activation of adenosine receptors and ADA .HIV gp120 prevents interaction between CD26, a costimulatory signal for TCR-mediated T cell activation, and ADA; this is implicated in the weak response of T cells to HIV . In addiOur treatment-naïve HIV patients had increased ADA levels, as previously described , and treAnother possible explanation for this maintenance of increased ADA level in TDF-treated patients is that under antiretroviral therapy, ADA response is affected by a previous depletion of CD4+ T cells .Moreover, in RA, serum ADA reflects monocyte/macrophage activity . Our datHIV produces chronic inflammation and immune activation that continues even in the presence of HAART and despite undetectable viral levels and viral latency, indicating that antiretroviral therapy is not able to completely restored the immune function ,20. In op = 0.231). In all treatments, we observed a non-significant decrease in IL10  had increased expression of this cytokine. Tenofovir, a structural analog of AMP, causes the inhibition of ATP transport through pannexin-1 , which cAll these data may explain why increased ADA levels were observed in TDF-treated patients despite the fact that the disease is apparently controlled by antiviral therapy. As mentioned previously, during latency the virus establishes survival mechanisms. Increasing ADA values might be one such mechanism, as an increase in this enzyme is related to a decrease in the immunomodulatory effect of adenosine, and sustained inflammation may be important to maintaining viral reservoirs. Therefore, as we know that tenofovir modulates ATP/adenosine metabolism, the inflammatory stage seen in our patients may be mediated by the antiviral drug by itself, and the increased ADA activity observed in TDF-treated patients compared to the other groups also indicates a regulation of the enzyme mediated by the drug. Further research must be conducted to understand the differences in the impact on ADA among tenofovir compositions, which may be related to the different half-life of TAF and TDF in blood.The chronic inflammatory state in our patients, together with the increase in ADA values in TDF-treated patients, may provide evidence of how one of the main comorbidities of HIV infection, decreased bone mineral density (BMD), could be regulated by tenofovir in early stages of antiretroviral therapy . AdenosiFinally, the possible role of adenosine deaminase acting on RNA (ADAR) must be taken into consideration, as these enzymes produce adenosine-to-inosine deamination in regions of the RNA, and play important roles during viral infections . ADAR caIn conclusion, ADA values in HIV patients can be used as a biomarker of the progression of the disease. Levels of ADA may be modulated by the antiretroviral therapy of choice, affecting the associated comorbidities . Tenofovir (concretely TDF) may potentiate both the inflammatory profile and the consequent degradation of adenosine by ADA and its associated deleterious effects.n = 21), (2) TAF  10 mg/day (n = 22), and (3) abacavir/dolutegravir/lamivudine in combination (Triumeq)  600 mg/day (n = 39). The protocol for this study was approved by the clinical research ethics committee (CEIm) of the Hospital Fundación Jimenez Diaz  and is in adherence with the tenets of the Declaration of Helsinki. All patients provided signed informed consent before being included in the study.We included 92 adult men recently diagnosed with HIV infection who were naïve to treatment at the initiation of the study. Patients were enrolled between May 2016 and September 2017 in the Infectious Disease Division in Hospital Fundación Jimenez Díaz, a teaching hospital in Madrid, Spain. Patients were grouped according to the antiviral treatment they received, the decision for which was based on medical criteria, i.e., (1) TDF  245 mg/day , diabetes, corticosteroid treatment, rheumatic diseases, renal failure, thyrotoxicosis, advanced liver disease, malabsorption syndrome, or neoplasias.A second limitation concerns the lack of a study group consisting of individuals not infected with the HIV virus; nonetheless, our results can be contrasted with well-established findings from studies conducted in the general population. Though our cohort consists of individuals who have lived with HIV infection for a short time, certain bias may have been introduced in this regard, as we cannot rule out a slight impact of HIV infection on metabolism in the first stages of infection. However, the fact that over 90% of patients had stage-A disease indicates an appropriate degree of homogeneity.Blood was collected at baseline and then at 3 and 12 months after the initiation of treatment under fasting conditions. ADA determination was performed using a kinetic colorimetric assay  based on the enzymatic deamination of adenosine to inosine in the Department of Clinical Analysis and Biochemistry at Hospital Fundación Jimenez Díaz. For humans, normal ADA reference values for serum and plasma range from 0 to 15 U/L. Counts of monocytes and neutrophils were obtained from the corresponding clinical records derived from studies of blood samples.One limitation of the study is the number of patient samples collected at each time point. For some procedures we were unable to obtain samples from all patients.® technology . The limits of detection were as follows: GM-CSF, 0.6 pg/mL; INFγ, 0.94 pg/mL; IL10, 0.93 pg/mL; IL12 (p70), 0.27 pg/mL; IL13, 0.34 pg/mL; IL1β, 0.24 pg/mL; IL2, 0.30 pg/mL; IL4, 1.84 pg/mL; IL5, 0.22 pg/mL; IL6, 0.17 pg/mL; IL7, 0.6 pg/mL; IL8, 0.25 pg/mL; and TNFα, 0.21 pg/mL. Intra-assay coefficients of variation for the cytokines studied were less than 5%; inter-assay coefficients of variation were less than 15% for both multiplex kits.We used the MILLIPLEX MAP Human High Sensitivity T Cell Magnetic Bead multiplex kit  to measure plasma concentrations of pro-inflammatory , INFγ (interferon gamma), GM-CSF (granulocyte-macrophage colony-stimulating factor), IL6, IL7, IL2) and anti-inflammatory cytokines . Samples were analyzed in duplicate following manufacturer recommendations. Briefly, after centrifugation (15 min at 2500× g), plasma aliquots were kept at -80°C until used. ELISA multiplex was done following manufacturer protocol, including beads preparation, standard and quality controls. Each sample was analyzed in duplicate. by LUMINEX MAGPIXt test, Chi square test, or Fisher’s exact test. Cytokines and changes in ADA are described as median values and quartiles (percentiles of 25% and 75%) and are compared using the Wilcoxon rank test. Correlation between variables was studied using Spearman’s rank correlation coefficient. For all determinations, we used R software version 3.6.0 (R Core team (2020); R Foundation for Statistical Computing, Vienna, Austria), and statistical significance was set at p < 0.05.Qualitative variables were expressed as frequencies and percentages, whereas quantitative variables appear as mean and standard deviation, or median and interquartile range, according to the results of a normality test (Kolmogorov–Smirnov test). Comparisons between quantitative variables were performed using Student’s