HIV/AIDS patients who fail to respond to first-line treatment protocols are switched to second-line ART. Identifying factors that influence effective second-line treatment can improve utilization of limited medical resources. We investigated the efficacy of long-term second-line anti-retroviral therapy (ART) after first-line virologic failure as well as the impact of non-nucleotide reverse transcriptase inhibitor (NNRTI), nucleotide reverse transcriptase inhibitor (NRTI), and protease inhibitor (PI) resistance mutations and medication adherence on ineffective viral suppression.+ T cell count, viral load, and drug resistance genotypes were quantified.A total of 120 patients were evaluated at 6, 12, 18, 24, and 48 months after initiation of second-line ART; a paper questionnaire was administered via a face-to-face interview and venous blood samples were collected. CD4+ T cell counts increased from 170 cells/μL (IQR 100–272) at baseline to 359 cells/μL (IQR 236–501) after 48 months of second-line treatment. Viral load (log10) decreased from 4.58 copies/mL (IQR 3.96–5.17) to 1.00 copies/mL (IQR 1.00–3.15). After switching to second-line ART, nine patients newly acquired the NRTI drug-resistant mutation, M184 V/I. No major PI resistance mutations were detected. Logistical regression analysis indicated that medication adherence < 90% in the previous month was associated with ineffective viral suppression; baseline high/low/moderate level resistance to 3TC/TDF was protective towards effective viral suppression.CD4Long-term second line ART was effective in the Henan region of China. Drug resistance mutations to NRTIs were detected in patients receiving second-line ART, suggesting that drug resistance surveillance should be continued to prevent the spread of resistant strains. Patient medication adherence supervision and management should be strengthened to improve the efficacy of antiviral treatment. In 2003, the “Four Free and One Care” antiretroviral therapy treatment (ART) policy was formally implemented in China . This po+ T cells in HIV-infected patients within 12 months of the switch to second-line treatment; HIV viral loads were lower than at the time of first-line failure in these patients as well [+ T cell counts were 531 after 36 months of second-line ART in patients from northeast China [+ T cell counts within 24 months of switching to second-line therapy [Although second-line treatment regimens include two NRTIs, 3TC and TDF, multidrug resistance and cross-resistance mutations acquired during first-line ART often results in only partial effectiveness of the NRTIs during second-line treatment. Some small sample size studies conducted in China have reported an increase in CD4 as well –11. Dingst China ; thus, l therapy –17. The  therapy . HoweverDrug selection pressure caused by the therapy regimen changes can lead to the accumulation of drug resistance mutations during long-term treatment. An increase in NRTI drug resistance was reported in patients after switching to the second line regimen . ProteasHere, we describe a prospective study of a cohort of patients with AIDS within 4 years of initiation of second-line treatment in Weishi County of Henan Province in China. We examined both the effectiveness of second-line treatment as well as associated factors which can impact treatment; importantly, we also analyzed whether cumulative NRTI resistance mutations acquired during first-line therapy failure affected the efficacy of second-line therapy. We also analyzed changes in PI, NRTI, and NNRTI resistance mutations after long-term second-line therapy.This research was approved by the Institutional Review Board of the first Affiliated Hospital of China Medical University. All participants signed informed consent statements.Patients were recruited from Henan between August 2009 and February 2010. Inclusion criteria were those listed in the Chinese National Free AIDS Antiretroviral Therapy Manual issued in 2009  and in t+ T cell count, HIV viral load, and “in-house” HIV genotype drug resistance test. During second-line therapy, “ineffective viral suppression” is defined as follows: plasma HIV-1 RNA level > 1000 copies/mL after 12 months of treatment; viral rebound which is defined as plasma HIV-1 RNA level > 1000 copies/mL after initial virologic suppression during second-line ART.After 6, 12, 18, 24, and 48 months of second-line therapy, we administered a paper questionnaire through a face-to-face interview and collected blood samples to assess patients’ CD4+ T lymphocyte count: a FACSCalibur flow cytometer  was used to enumerate CD4+, CD8+, and CD3+ T lymphocytes. Peripheral venous blood was collected and treated with EDTA-3 K anticoagulant; T lymphocyte counts were obtained within 24 h.CD47 copies/mL.Viral load: Plasma obtained from patients’ venous blood samples was stored at − 80 °C until testing. HIV viral load was determined using a COBAS AmpliPrep/COBAS TaqMan HIV-1 test . The detection range of the COBAS Assay for viral load quantification is 20–10HIV-1 viral RNA was extracted from 200 μl plasma with a QIAamp Viral RNA Mini Kit . One-step Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using a TaKaRa One-step RNA PCR Kit . The HIV-1 pol gene was amplified using first round primers MAW26  and RT21 ; amplification was achieved using 1 cycle of 50°C for 30 min, 1 cycle of 94 °C for 5 min, and 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min 30 s, with a final extension of 72 °C for 10 min in the first round; and second round primers PRO-1  and RT4R ;amplification was achieved using 1 cycle of 94 °C for 5 min and 30 cycles of 94 °C for 30 s, 63 °C for 30 s, and 72 °C for 2 min 30 s, with a final extension of 72°Cfor 10 min [pol gene; negative control: specimens that are HIV-negative; blank control: amplification without template. The negative and positive controls were extracted, amplified, detected, and analyzed simultaneously with the research sample. Sequences were aligned using Contig software and edited using Bioedit software. The resulting sequences were submitted to the Stanford University HIV drug resistance database (http://hivdb.stanford.edu) for interpretation of putative drug resistance results.Drug-resistant mutation analysis: Mutations in the HIV polymerase gene, Pol, were screened for drug resistance mutations. r 10 min , 26. PosP < 0.05 for two-sided tests was defined as statistically significant.SPSS software (version 17.0) was used to analyze quantitative data. Categorical data was described by rate or ratio and analyzed by either Chi-square test or Fisher’s exact test. Continuous data was described by the mean and standard deviation if data met the hypothesis of normal distribution, otherwise median and inter-quartile ranges (IQRs) were used. Univariate and multivariate logistic regression were performed to identify possible associated factors that may have contributed to viral suppression. + T cell count  were 4.58 (3.96–5.17) copies/ml and 170 (100–272) cells/μl, respectively. After first-line failure, drug resistance rates to 3TC and TDF were 32.5% (38/117) and 48.7% (57/117), respectively  years, 60.8% (73/120) were male, 39.2% were female (47/120). 95.8% (115/120) of patients were infected through paid blood donation(blood transfusion). All HIV Pol sequences were subtype B interpreted by the Stanford University HIV drug resistance database. All 120 patients used first-line therapy regimens (2NRTIs + 1NNRTIs); first-line treatment duration was 5.0 (3.7–5.5)  years. At initiation of second-line therapy, patient baseline viral load (lg10)  and mean CD4Staff members regularly followed up with patients. After switching to second-line treatment, 102, 104, 82, 90, and 85 patients were retained for follow-up at 6, 12, 18, 24, and 48 months, respectively. Of the 16 patients who were lost to follow-up at 6 months, two continued second-line therapy according to drug receipt records from the CDC; however, we were unable to collect blood samples and therapy records from these two patients as they were away from Weishi county, Henan province at 6 months. We were able to retrieve blood samples and therapy information from these patients at 12 month. The number of patients who were lost to follow-up increased to 30 at 18 months; Another eight patients continued second-line therapy according to drug receipt records from the CDC; however, we were unable to collect blood samples and therapy records from these patients as they were away from Weishi county at the 18 month timepoint. We were able to obtain their blood samples and therapy information at the 24 month timepoint. Finally, 35 patients were lost to follow-up at 48 months.+ T cells increased from a baseline of 170 cells/μL (IQR 100–272) at initiation of second-line therapy to 359 cells/μL (IQR 236–501) after 48 months of treatment, which was statistically significant (P < 0.05) .During long-term second-line therapy, patients’ CD4 IQR 100–72 at ini10) decreased from an average baseline level of 4.58 copies/mL (IQR 3.96–5.17) to 1.00 copies/mL (IQR 1.00–3.15) after 48 months of second-line therapy. HIV viral load decreased significantly (P < 0.05) after several different durations of second-line treatment (Table P < 0.001). The proportion of patients with viral loads < 1000 copies/mL at 6, 12, 18, 24, and 48 months was 61.8% (63/102), 71.1% (74/104), 69.5% (57/82), 70.0% (63/90), and 75.3% (64/85), respectively. The proportion and frequency of patients with < 50 HIV-RNA copies/mL at 6, 12, 18, 24 and 48 months was 43.1% (44/102), 43.4% (45/104), 42.7% (35/82), 50% (45/90), and 67.1% (57/85), respectively.Viral load (lognt Table . The prosecond-line treatment were HIV subtype B as interpreted by the Stanford University HIV drug resistance database. Rates of drug resistance to NRTIs were 58.1% (68/117), 43.3% (13/30), 31.8% (7/22), 28.0% (7/25), 18.1% (2/11), 31.6% (6/19) at baseline, 6, 12, 18, 24, and 48 months, respectively. Rates of drug resistance rates to NNRTIs were 71.8% (84/117), 56.7% (17/30), 45.5% (10/22), 24.0% (6/25), 36.3% (4/11), and 36.8% (7/19) at baseline, 6, 12, 18, 24, and 48 months, respectively. No major resistance to PIs was detected in our study; minor drug resistance to the PIs, A71V/T and L10I, were detected during second-line therapy.In-house genotype drug resistance tests were performed on samples from patients whose viral loads were > 1000 copies/ml. All sequences obtained during After switching to second line ART, newly acquired NRTIs resistance mutations at position 184, which are associated with resistance to 3TC, were detected in nine HIV/AIDS patients. The M184 V/I mutation was detected in five patients, one patient, two patients, and one patient at the 6, 12, 18, and 48 month timepoints, respectively. Mutation K65R, associated with resistance to TDF, was found in four patients at baseline; however, after switching to second-line ART, K65R mutations were not detected. TAM  and D67N mutations, associated with resistance to TDF, were detected during second-line ART. NRTI drug-resistance mutations were not found in some patients from the baseline point to 48 months after beginning second-line treatment  medication adherence (the past month) below 90% (vs. 90–100%) ; 2) compliance unknown (vs. 90–100%) , these two factors were protective toward ineffective viral suppression. 3) baseline high level resistance to 3TC/TDF (vs. sensitive) ; and 4) baseline low/moderate level resistance to 3TC/TDF (vs sensitive)   of patients achieved viral suppression within 24 months in our study, which was similar to results reported from South Africa in which CD4+ T counts increased by an average of 177cells/L and 75% (74/99) of patients achieved a viral load < 1000 copies/mL within 24 months of beginning second line therapy [+ T counts increased to 189 cells/L and 75.3% (64/85) of patients achieved a viral load < 1000 copies/mL. These results indicate that long-term treatment was effective in our cohort.In our study, patients were recruited from Henan province in China; Henan was one of the earliest areas to begin free first-line ART for HIV-infected citizens and was the first to initiate second-line antiretroviral therapy as described in the National Free Antiretroviral Treatment Program (NFATP). During the 4 years of follow-up, patient CD4 therapy . We alsoDrug-resistance mutations were detected when the viral load reached > 1000 copies/ml during second-line ART follow-up; yet, our results show that newly acquired NRTI resistance mutations were few. The M184 V/I mutation was detected in nine patients after switching to second-line therapy, which can attributed to the use of 3TC in the program. Boyd et al. found that one patient newly acquired M184 V resistance mutations after 96 weeks second-line ART . TDF wasMajor PI resistance mutations were not detected using the “in-house” genotype drug resistance test. However, we obtained HIV Pol sequences using monoclonal methods; the results of a rootless radial tree generated via phylogenetic analysis at the two time points (baseline and after 48 months of second-line treatment) showed significant evidence of viral evolution, suggesting that resistance surveillance should be continued.After switching to the second-line regimen, 31 patients exhibited increased viral load; of these, 28 patients exhibited viral loads > 1000 copies/ml after at least 12 months of second-line therapy. We performed a logistical regression analysis of the data; our results indicated that medication adherence (in the past month) below 90%, compliance or when medication adherence was unknown was associated with ineffective viral suppression. These results are consistent with the work reported by Murphy et al. . In a stIn our cohort, baseline resistance to 3TC/TDF resulted from ineffectiveness of the second-line ART; which was similar to the research reported regarding second line treatment in Africa , 31. PatLong-term second-line antiviral therapy in the Henan region was effective. During the four years of second-line therapy, drug mutations conferring resistance to NRTIs were observed among patients receiving second-line ART, although major drug resistance mutations were not detected in the PI region. Drug resistance monitoring should be continued to prevent the spread of resistant strains. In addition, medication adherence supervision and management should be increased in order to improve the effectiveness of antiviral drugs.


The study aims to elucidate the spectrum, magnitude and determining factors of the major opportunistic infections in PLHIV patients currently receiving HAART.A retrospective cross-sectional study was conducted at Tercha Hospital from 744 patient cards. The overall all prevalence of opportunistic infection was 658 (88.4%) developed OIs. Pulmonary tuberculosis, 118 (18%), severe community acquired pneumonia 107 (16.3%) and oral candidiasis 103 (15.6%) were the most common opportunistic infections. Disease stage [AOR = 3.22:95% CI 1.76–5.66], CD4 level [AOR = 2.53:95% CI 1.19–5.37], drug adherence [AOR = 3.02:95% CI 1.57–5.77] and hemoglobin [AOR = 2.49:95% CI 1.34–4.62] showed significant association with OIs. Higher magnitude of opportunistic infection with considerable proportion of AIDS defining illness was detected. So empowerment of skilled man power, health education and provision of antimicrobials is mandatory.The online version of this article (10.1186/s13104-018-3707-9) contains supplementary material, which is available to authorized users. Human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS) is the world’s most serious health and development challenge, affecting approximately 36.9 million people . EthiopiHIV became responsible for significant morbidity and mortality due to underlying immune-suppression which leads to life threatening opportunistic infections (OIs) during the natural course of the disease . ScientiSince the advent of highly active antiretroviral treatment (HAART), the incidence of OIs in people living with HIV/AIDS, (PLHIV) reduced . HoweverOpportunistic diseases like Candida esophagitis, Pneumocystis Carinii pneumonia (PCP), active pulmonary tuberculosis, Mycobacterium avium complex (MAC) infection, cytomegalovirus (CMV), Cryptococcal meningitis (CRM), Kaposis sarcoma (KS), and herpes zoster were prevalent in HIV patients . In EthiOpportunistic infections lead to frequent morbidity and mortality which shortens the life span of people with HIV infections and requires expensive treatments which becomes a burden for a developing country .Timely intervention of OIs not only helps HIV positive persons to live longer but it also helps to prevent transmission spreading to others in the community . Since pThe study was conducted at Tercha Referral Hospital situated at Tercha Town which is a capital city of Dawro Zone in South Ethiopia. It is serving a population of about 2 million people coming from different nearby zones and woredas. It also provides care and treatment for HIV/AIDS patients including ART , voluntary counseling test, provider initiative counseling and testing. The hospital provides inpatient and outpatient services including ART services. As of 2013, the Federal ministry of health, Ethiopia adopted the 2013 World Health Organization recommendation, that all HIV positives are eligible for ART. The ideal time for ART initiation depends on the clinical condition and readiness of the client. All PLHIV start ART as early as possible to all adults with a confirmed HIV diagnosis ready and willing regardless of their WHO clinical stages and CD4 counts .Institution based cross-sectional study design was conducted from May 1 to August 30 2017.All people living with HIV/AIDS (PLHIV) ≥ 18 years old currently using HAART.Nine hundred sixty-four patients were following their routine treatment in the hospital, of these 87 of them were children aged < 18 years. Thirteen of them were lost to follow up, 19 of them dropped from their course of treatment and 65 PLHIV were transferred to another health care facility. Incomplete data were obtained from 36 patients. Finally, 744 patients who are on ART follow up taking HAART from 2013 to 2017 were our sampling participants.Adherence defined as good if adherence is > 95% (< 2 doses of 30 doses or < 3 dose of 60 dose is missed); poor if adherence is between 85 and 94% (3–5 doses of 30 doses or 3–9 dose of 60 dose is missed). Adherence was measured by pills count.Lost to follow up Not seen since ≥ 1 month < 3 months.Dropped Lost to follow up for > 3 months.SCAP is defined is acute infection of the pulmonary parenchyma, and patients will often present with multiple organ failure with symptom onset in the community within 48 h of hospital admission.Transferred A patient is referred to another health facility for care evidenced by his/her document.Data were collected using a standard checklist prepared in English, retrieved from the hospital’s ART registry document which is a standard format for sending comprised data to health monitoring information system. Socio-demographic variables, clinical information, type of OIs, prophylaxis usage, WHO clinical staging, CD4 count, hemoglobin level, and weight were collected from patent charts by experienced nurses. Patients’ clinical records that were not complete or were missing data were omitted. The data collection format was checked for its completeness and consistency with the patient’s clinical records by a supervisor and the investigators on a daily basis. Five nurses working in other government health centers were involved in the data collection procedure.Data were entered and cleaned in Epi-info version 3.5 and analyzed using SPSS version 20. Bivariate analysis was conducted primarily to check association of each variable with OIs. Variables found to have association with the dependent variable at P-value of 0.2 in the bi-variate analysis was entered into multiple logistic regression model. The variables that showed significant association with P-value < 0.05 in the multivariate logistic regression was independent factors.Out of 744 PLHIV, 443 (59.5%) were females and 254 (54.5%) were found in the age group of 18–30 years. Regarding to religion, majority of them 347 (46.6%), were orthodox. Among the participants, 587 (78.9%) of them were jobless and 432 (58.1%) were married. Urban dwellers in this study were 507 (68.1%).  were using alcohol, 11 (1.4%) were smoking cigarette, and 8 (1.1%) were chewing kchat. Regarding to the current status of the patients, majority of them 597 (80.2%) were in WHO stage I. In case of CD4 count, 381 (51.2%) of them had CD4 count of < 200. Concerning hemoglobin, 182 (24.5%) of them having < 10 g/dl and 371 (49.8%) were having < 60 kg weight. Most of them 592 (79.6%) had not been using condom and 606 (81.4%) of the individuals had good HAART adherences  developed OIs, of which 232 (35.2%), 157 (23.9%), 146 (22.2%), and 123 (18.7%) developed 2, 3, ≥ 4, and 1 OI respectively. The spectrum of OIs was ranging from the common oral candidiasis to the life threatening CNS toxoplasmosis and the fungal cryptococcal meningitis. Pulmonary tuberculosis, 118 (18%) was the most common OI. Next to TB, Severe community acquired pneumonia (SCAP) 107 (16.3%) and oral candidacies 103 (15.6%) were the most common OIs in this study. Only five and 2 individuals were affected by Toxoplasmosis and KS respectively  of them died of the OI. The main cause of death were bacterial meningitis which accounts 16 (28.6%) and pulmonary TB and SCAP accounted for 13 (23.8%) of death each  which is consistent with a study conducted in Cameroon 25%  and 31% Oral candidiasis prevalence (1.6%), which is the third most common OI in this study, corroborated with 16.4% prevalence rate in Zewditu memorial hospital . In the AIDS defining illness, like PCP, CRM, cryptosporidiosis and KS were identified in this study. Magnitude of PCP 3.9% observed in this study was higher than 2.1% in Zewditu memorial hospital , 1.5% prCryptosporidiosis finding of the present study 9.1% was corroborated with 13% prevalence detected in southern Ethiopia  but highGood HAART adherence became a predictor for the occurrence of OIs in this study which was corroborated with studies conducted in North western Ethiopia , NigeriaHuman immunodeficiency virus infection is frequently associated with anemia where patients with more advanced HIV disease or a lower CD4 cell count had higher rates of anemia. Severe anemia was associated with a much faster rate of HIV disease progression and confirmed that anemia is a strong independent predictor of death. In different study settings, the prevalence of anemia has been estimated to be 30% in patients with asymptomatic HIV infection and 63–95% in persons with AIDS . Hemoglo3 were found to be an independent predictor for developing OIs. This finding is in complete agreement with studies conducted in Gondar and India which reported high risk of developing OIs among this group [CD4 count < 200/mmis group –19. ThisAdvanced baseline WHO stage was also another factor associated with OI in this study which was also observed in different studies conducted in the country and elsewhere , 34, 35.The overall prevalence of OI in the era of HAART is higher as compared with previous studies in the country. Significant level of AIDS defining illness was noticed. WHO stage II–IV, CD4 level, ART adherence and hemoglobin level became predictors for the occurrence of OIs. So skilled health professionals for proper diagnosis and management of OIs are mandatory and provision of adequate and sustainable antimicrobials and close monitoring and follow-ups are of critical importance.The retrospective nature of the study makes some the finding less informative and as a cross sectional study, cause-effect relationships cannot be assessed.Additional file 1: Table S1. Frequency distribution of socio-demographic characteristics of PLHIV (n = 744) in Tercha Hospital in 2017. Table S2. Frequency distribution of outcomes of OIs of PLHIV in Tercha Hospital in 2017.


For people living with HIV (PLWH), antiretroviral therapy (ART) adherence is crucial to attain better health outcomes. Although research has leveraged consumer health information technologies to enhance ART adherence, no study has evaluated feasibility and clinical outcomes associated with the usage of a commercially available, regularly updated mobile health  app for improving ART adherence among PLWH.This study aimed to assess the feasibility, acceptability, and clinical outcomes of Care4Today, an existing, free, biprogrammatic mHealth app for improving ART adherence among PLWH.The Florida mHealth Application Adherence Project (FL-mAPP) was a 90-day longitudinal pilot study conducted in 3 public HIV clinics in Florida, United States. After obtaining informed consent, 132 participants completed a survey and then were given the option to try an existing mHealth app to help with ART adherence. Of these, 33.3% (44/132) declined, 31.1% (41/132) agreed but never used the app, and 35.6% (47/132) used the app. All were asked to complete follow-up surveys at 30 days and 90 days after enrollment. Usage data were used to assess feasibility. Clinical outcomes of self-reported ART adherence and chart-obtained HIV viral load and CD4+ T-cell counts were compared among those who used the platform (users) versus those who did not (nonusers). Participants and HIV care providers also provided responses to open-ended questions about what they liked and did not like about the app; comments were analyzed using thematic analysis.P=.12). Overall, 39 (82%) users liked or somewhat liked using the platform. Participants’ favorite features were medication reminders, ability to create custom reminders, and adherence reports.Of 132 participants, 47 (35.6%) and 85 (64.4%) were categorized as users and nonusers, respectively. Among users, a Kaplan-Meier plot showed that 25 persons (53%) continued using the app after the 90-day follow-up. At 30-day follow-up, 13 (81.3%) of those who used the mHealth app reported ≥95% ART adherence, compared with 17 (58.6%) nonusers (This longitudinal study found that a commercially available medication adherence mHealth app was a feasible and acceptable intervention to improve ART adherence among PLWH and engaged in clinical care across 3 public HIV clinics in the state of Florida. Overall, participants liked the Care4Today app and thought the medication reminders were their favorite feature. Generally, self-reports of ART adherence were better among users than nonusers, both at 30- and 90-day follow-ups. Further clinical research needs to address user fatigue for improving app usage. Although up to 85% of PLWH in the United States repordherence , concerndherence -10. Althdherence -25 and mdherence -31, no sdherence .Although 3 meta-analyses -35 foundCare4Today Mobile Health Manager (Johnson & Johnson) is a commercially available mHealth app designed to improve medication adherence and has been described in detail elsewhere . The appThe objective of this study was to assess the feasibility and acceptability of Care4Today among PLWH. More specifically, we sought to evaluate (1) app usage pattern; (2) changes in self-reported ART adherence, HIV viral load, and CD4+ T-cell count among PLWH who used the app compared with those who did not; and (3) preferences and suggestions for app features for PLWH. As a secondary goal, we sought to assess the interest of HIV providers and clinical staff in receiving app-generated adherence data.The Florida mHealth Application Adherence Project (FL-mAPP) was a 90-day feasibility and acceptability longitudinal pilot study of a commercially available mHealth app for treatment adherence among PLWH, conducted from 2015 to 2016. As previously described, participants were recruited from public HIV clinics  affiliated with the Florida Department of Health . EligibiAfter providing informed consent, participants completed a survey questionnaire. They were then able to choose by trying the app in 2 forms: (1) SMS reminders only (for flip phone owners) or (2) full app (for smartphone owners). Clinic personnel assisted participants as needed with downloading the app, creating their account, and configuring the app or SMS reminders. All participants were asked to complete 2 follow-up surveys at 30 and 90 days after enrollment. Participants who chose to try the app or SMS reminders were asked if we could submit a dashboard-generated adherence report to be reviewed by their provider as part of ongoing clinical care. All of these participants accepted the request.Participants received gift cards totaling US $30 over the course of the study in compensation for their time completing the surveys regardless of the decision to use the app. Follow-up clinical measures included 30- and 90-day self-reported ART adherence, HIV viral load, and CD4+ T-cell count.Surveys were conducted at baseline, interim (day 30), and endpoint (day 90). Baseline paper surveys were completed in person at the clinics, and follow-ups were conducted either in person or over the phone by research staff, as described previously . The basParticipants reported their age in years , gender assigned at birth, race and ethnicity , highest education level attained , and marital status .nonusers, and those who used the app at least once were defined as users.We assessed usage of the app at 30 and 90 days after enrollment among those who used the SMS or app at least once. For each participant, continued use was defined as number of days elapsed from enrollment until the last time the participant reported either a taken or a missed dose via the app or SMS. For analysis, persons who declined to try the app and those who agreed to try but never actually used it were categorized together as Over the last 30 days, how many days did you forget or miss your HIV medications, even partially? We categorized participants’ ≥95% adherence if they reported missing doses on less than 2 days in the previous month. We also attempted to define ART adherence according to the responses from the app, but if participants did not report on a specific day, it was not possible to tell if they were nonadherent or just forgot to enter information into the system.We measured self-reported ART adherence using a previously developed item that was validated for PLWH . ParticiWe collected HIV viral load and CD4+ T-cell count at 2 distinct intervals: (1) baseline viral load and CD4+ T-cell count at enrollment day or the 30 days preceding enrollment and (2) the next available medical record laboratory results after enrollment. We used a plasma level cut-off point of less than 40 copies/ml of HIV-1 RNA to define optimal viral suppression.How did you like using the Care4Today system? What stopped you from using the system more? Which features did you like the most? What did you not like about Care4Today? How can the system be improved?At study day 30, participants who chose to try the app answered open-ended questions that assessed acceptability . Items iHow interested would you be in continuing to receive information on patients’ self-reported adherence with this dashboard?How often would you like to see these type of data about your patients? and How interested would you be in learning how to access the dashboard online to look at adherence reports of your patients who are using the “Care4Today” app?Participant providers were invited to participate in the study by receiving printed copies of app-generated adherence reports at day 30, 60, and 90. After day 90 of the study, consenting providers completed brief surveys regarding their interest in receiving the app-generated adherence data. Items included the following: For quantitative data, proportions and chi-square tests compared baseline demographic characteristics, ART adherence, and clinical variables between users versus nonusers , both atFor responses to open-ended questions, we used an inductive thematic analysis  to unveiAll participants were provided with written informed consent, and Health Insurance Portability and Accountability Act required health authorization to use personal health information for research. The institutional review boards of the Florida Department of Health, the University of South Florida, and the University of Florida reviewed and approved all procedures and data management processes.users). Nonusers  comprised 44 participants  who declined to try the app and 41  who agreed to try the app but did not use it. Of 47 Users, 37 (78%) who chose to use the full smartphone app and 10 (21%) who chose SMS. We conducted an analysis to compare these 2 groups and found no significant differences other than smartphone ownership, which was significantly more frequent among those who chose to use the full app. At baseline and follow-up, no significant differences were found in ART adherence, HIV viral load, or CD4+ T-cell count between users and nonusers  between ≥95% adherence reported by app users  compared with just over half of nonusers . Similarly, at 90-day follow-up, 7/15 users (46%) reported ≥95% ART adherence compared with 9/22 nonusers (40%), although this difference was not significant.From baseline, 81% (38/47) of app users and 64% (54/85) of nonusers completed 90-day follow-up surveys. Overall, at 30-day follow-up, there were 47 users who used the app at least once, of whom 39/47 (82%) liked or somewhat liked using the app, 1/47 (2%) did not like it, and 7/47 (15%) did not respond the question. Favorite features were medication reminders (n=25), ability to create custom reminders (n=5), adherence reports (n=5), and multiple features (n=4). Open-ended questions identified app features and technical issues that participants found challenging. The qualitative findings are available in A total of 10 clinicians completed the survey: 5 nurse practioners and 5 physicians. The average age was 47.8 years (SD 15.6) with the majority being female  and white non-Hispanic . Of the 10 providers, 6 had over 10 years of experience working with PLWH, 1 had 6 to 10 years, and 3 had 5 years or less. Although 90% (9/10) of providers were either very or somewhat interested in reviewing adherence reports through the app, 80% (8/10) wanted to see these reports either monthly or quarterly (as opposed to daily or weekly). Finally, 5 providers (3 nurse practitioners and 2 physicians) had low to no interest in logging in to the provider interface to access the reports.This longitudinal study found that a commercially available medication adherence mHealth app was a feasible and acceptable intervention to improve ART adherence among PLWH engaged in clinical care across 3 public HIV clinics in the state of Florida. To our knowledge, this is the first feasibility pilot study that used a free, commercially available mHealth app to improve medication adherence among PLWH. Overall, participants liked the Care4Today app and favored the medication reminders feature. Generally, self-reports of ART adherence were better among users than nonusers, both at 30- and 90-day follow-ups, although the difference between groups was not statistically significant. Given the main goal of this study was to assess acceptability and feasibility of the intervention, we did not intend to demonstrate statistical significance of our findings. Rather, we sought to identify trends and factors that might inform a larger efficacy study. Importantly, although our findings do not support larger implementation of commercially available medication adherence apps for PLWH at this time, we did identify promising results in clinical outcomes that are commonly accepted proxies for efficacy .Among participants who had less than optimal ART adherence at baseline, over half of them started and kept using the intervention for the entire duration of the study. Although the drop-in usage between 30- and 90-day follow-ups might seem considerable when compared with previous research , some clOf our participants with less than optimal ART adherence at baseline, the proportion of persons who reported good ART adherence at follow-up was higher among app users than nonusers. Although this difference was not statistically significant, this finding suggests that use of the app did improve medication taking, and this could be assessed with a larger clinical trial. Although self-reported ART adherence has been consistently associated with improved clinical outcomes, including viral suppression ,49, mostParticipants’ feedback focused on suggestions to improve features and technical aspects of the app. Among these, suggestions about personalized and customized reminder characteristics were paramount. Moreover, users wanted an app that would work even if they were off a wireless internet connection. These recommendations speak about the user’s desire of more independence when using mHealth apps. However, these requests need to be balanced with the need to keep apps efficient and fun to use. Potentially, these objectives can be met with commercially available apps that are developed and maintained by dedicated personnel in charge of updating and enhancing app databases and features. On the contrary, providers’ feedback showed clinicians see value in accessing self-reported adherence data from their patients during clinical visits. This seems to fit well with the clinical workflow; visits provide the opportunity to address adherence inconsistencies that may be reported through the mHealth app reports. However, in order for providers to retrieve these data on their own, mHealth apps should be able to easily communicate with information systems already being used by health professionals. Clinicians had less interest in viewing these reports on a daily or weekly basis. One potential way to link these reports into a workflow could be to establish a critical number of missing doses that would trigger an increased frequency of medication reminders, or suggestions for tele-consultations with clinical staff in advance of the next visit to address adherence inconsistencies promptly.users and nonusers, we were limited in our ability to conduct subgroup analyses at this time. These would be valuable to further contextualize the app’s utility in subpopulations of PLWH, and future research on commercially available medication adherence apps should consider assessing these differences. We noticed that almost 50% of participants had user fatigue throughout this 90-day pilot study. Active updates and app maintenance and a more robust contingency system (such as gamification) might help to sustain novelty and address this concern. ART adherence data relied on self-report. Future research should include not only a larger sample and experimental design, but also an objective measure of medication adherence.Our work has several limitations. This study was conducted at 3 HIV clinics affiliated to the Florida Health Department. Thus, our findings cannot be generalized to sites that operate with different procedures for patient engagement and follow-up. However, our work is of interest given that these clinics draw from a distinct socioeconomic group of diverse backgrounds. Almost two-thirds of study participants were categorized as app nonusers, either because they declined to try the app at all, had problems downloading and using the app, or did not use it after downloading. As reported previously , interesIn summary, Care4Today Mobile Health Manager, a commercially available, biprogrammatic medication adherence app that operates with both iOS and Android operating systems, as well as SMS, was found feasible and acceptable for improving ART adherence among PLWH during a 3-month longitudinal study. Further clinical research needs to address user fatigue to improve platform usage.


In low- and middle-income countries (LMICs), a substantial unmet need for affordable single-tablet regimen (STR) options remains. Rilpivirine  is formulated in a low-cost STR with tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC).Switching at Low HIV-1 RNA into Fixed Dose Combinations (SALIF) compared RPV with efavirenz (EFV), both as STRs with TDF and FTC, in maintaining virologic suppression.N = 426), stratified by NNRTI use, were randomised 1:1 to receive TDF/FTC/RPV (300/200/25 mg qd) or TDF/FTC/EFV (300/200/600 mg qd). Primary endpoint was proportion of patients with virologic suppression (HIV-1 RNA < 400 copies/mL) at week 48 .SALIF was a phase 3b, randomised, open-label, non-inferiority study in virologically suppressed adults (HIV-1 RNA < 50 copies/mL) on non-nucleoside reverse transcriptase inhibitor (NNRTI)-based first-line antiretroviral therapy (ART) in Cameroon, Kenya, Senegal, South Africa, Uganda and Thailand. Patients (n = 213) or TDF/FTC/EFV (n = 211). At week 48, virologic suppression was maintained in 200/213 (93.9%) patients in the RPV arm and 203/211 (96.2%) in the EFV arm , demonstrating non-inferiority of TDF/FTC/RPV. One patient in each arm experienced virologic failure without treatment-emergent resistance. Twenty-seven patients discontinued prematurely (8.0% RPV vs. 4.7% EFV), the most frequent reasons being adverse events , site closure (1.9% vs. 0.5%), loss to follow-up (0.9% vs. 1.4%) and consent withdrawal (0.9% vs. 1.4%).Patients received TDF/FTC/RPV (In adults with suppressed viral load on first-line NNRTI-based ART in LMICs, switching to an STR of TDF/FTC/RPV was non-inferior to TDF/FTC/EFV in maintaining high rates of viral suppression with a comparable tolerability profile. In low- and middle-income countries (LMICs), there is a substantial unmet need for affordable STR options. The WHO policy brief from July 20184 stated that dolutegravir (DTG)-based regimens may be recommended as a preferred first-line regimen for people living with HIV initiating ART, and the alternative first-line treatment regimen is efavirenz (EFV)-based. These first-line recommended treatments may result in some patients experiencing neuropsychiatric events or other tolerability issues,7 while the use of nevirapine (NVP) is associated with the risk of hepatotoxicity and skin reactions.8 Given that DTG is not recommended for use in non-pregnant women who are trying to conceive or during the first trimester of pregnancy because of concerns about a possible increased risk of neural tube defects,9 a viable alternative to DTG is needed. There is, thus, an unmet need for additional efficacious, well-tolerated and more affordable alternative ART regimens, particularly in LMICs.10Current HIV treatment guidelines11 and convenient once-daily dosing, with a reduced special effort access price of US$40 per patient per year in sub-Saharan Africa and least developed countries,12 making it a good candidate component for an affordable STR in LMICs.17 RPV, in combination, is indicated in treatment-naïve patients 12 years of age and older with a viral load of HIV-1 RNA ≤ 100,000 copies/mL.18 Tenofovir disoproxil fumarate (TDF)/lamivudine (3TC) or emtricitabine (FTC) with RPV is listed as a ‘preferred’ first-line regimen in European guidelines,3 has recently been adopted as a preferred first-line regimen in South African guidelines19 and is a recommended ‘alternative’ regimen in the United States and Thailand.20Rilpivirine  is a second-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) with a good safety profile21 In a 48-week pooled analysis of these trials, RPV was non-inferior to EFV both in patients with viral loads ≤ 100 000 copies/mL  and with viral loads > 100 000 to ≤ 500 000 copies/mL ,21 but non-inferiority was not achieved for patients with viral loads > 500 000 copies/mL . In addition to these pivotal trials using the individual agents, the STR of TDF/FTC/RPV has been evaluated in both treatment-naive patients and virologically suppressed patients, including in at least one LMIC setting. In these studies, TDF/FTC/RPV was found to be non-inferior to several different ART regimens, including protease inhibitor (PI)- and NNRTI-based combinations.25 Hence, in Europe and the United States, TDF/FTC/RPV is indicated for use in treatment-naive patients with HIV-1 RNA ≤ 100 000 copies/mL and for patients with suppressed viral load for ≥ 6 months prior to switching therapy and without known resistance-associated mutations to NNRTIs, TDF or FTC.27Approval of RPV was based on findings from two double-blind, placebo-controlled, phase 3 trials, ECHO and THRIVE, comparing RPV with EFV, most commonly in combination with TDF and FTC, in treatment-naive patients.28 Pharmacokinetic studies of RPV in pregnancy reveal that most women achieve effective plasma concentrations of RPV.31 The Antiretroviral Pregnancy Registry showed no increased risk in birth defects after first trimester RPV exposures as of January 2018.32 Furthermore, according to both the US Department of Health and Human Services Recommendations for Use of Antiretroviral Drugs in Pregnant HIV-1-Infected Women and the 2017 European AIDS Clinical Society guidelines, there are sufficient data from use in pregnancy to recommend RPV as an alternative agent in ART-naive pregnant women with viral loads ≤ 100 000 copies/mL and CD4+ counts > 200 cells/mm3.33Preclinical studies of RPV showed no evidence of teratogenicity RPV and therefore may be an option in populations with large proportions of HIV-infected women of childbearing potential who have access to regular viral load monitoring.This study was, therefore, designed to examine the utility of switching to the STR of TDF/FTC/RPV in LMIC patients with suppressed viral loads, who were on an NNRTI-based first-line ART.Switching at Low HIV-1 RNA into Fixed Dose Combinations (SALIF) was a 48-week, multicentre, phase 3b, randomised, open-label study designed to demonstrate non-inferiority of RPV to EFV (both coformulated with TDF and FTC) in maintaining HIV-1 RNA suppression (defined as HIV-1 RNA < 400 copies/mL) among adult patients in LMICs on first-line NNRTI-based ART (with EFV or NVP) with HIV-1 RNA < 50 copies/mL. The study was conducted at 23 sites in Cameroon, Kenya, Senegal, South Africa, Uganda and Thailand from 23 August 2013 to 27 October 2015.35Ethics committee approval was obtained at all participating centres in accordance with the principles of the 2008 Declaration of Helsinki. Central randomisation was based on a computer-generated schedule prepared before the study by the sponsor. Randomisation was balanced by using randomly permuted blocks and stratified by baseline NNRTI. A pre-specified interim analysis was performed once all patients had reached week 24 or discontinued earlier, and was reviewed by an independent data monitoring committee. Data from this study have been presented previously.All patients remained on study until the last patient reached the week 48 visit. Patients were then switched to an investigator-selected treatment according to local prescribing practice. In countries where a RPV-based regimen was not yet available, patients with suppressed HIV-1 RNA levels receiving TDF/FTC/RPV could continue in post-trial access programmes until RPV was publicly available in the country.36 a CD4+ cell count of more than 200 cells/mm3, and a preference to change their current ART for reasons of simplification and/or NRTI toxicity. Patients also needed to have access to at least one meal a day and have a normal electrocardiogram (ECG) to be eligible. Patients co-infected with Mycobacterium tuberculosis, who were likely to require rifampicin-based treatment during the study, were excluded. Written informed consent was obtained from each patient prior to the screening procedures.The study included adults  with documented HIV-1 infection, who had been receiving first-line NNRTI-based ART (defined as two nucleoside reverse transcriptase inhibitors [NRTIs] with either EFV or NVP) for at least 1 year, and the same ART for at least 8 weeks, before screening. Previous changes in NRTI background regimen were allowed, but patients who had previously switched from EFV to NVP for toxicity reasons were not eligible. At screening, eligible patients had to have suppressed viral loads, commonly accepted to be a plasma HIV-1 RNA < 50 copies/mL,At baseline, patients were randomly assigned (1:1) to receive an STR of either TDF (300 mg)/FTC (200 mg)/RPV (25 mg) or TDF (300 mg)/FTC (200 mg)/EFV (600 mg). Both products were supplied by the sponsor and given in accordance with the product labels at the recommended dose of one tablet per day. Patients randomised to TDF/FTC/RPV were advised to take the medication with food, whereas patients randomised to TDF/FTC/EFV were advised to take it on an empty stomach at bedtime. To assess adherence, patients were asked to bring the study drug containers, whether empty or not, to each study visit.me HIV-1 RNA assay with a lower limit of quantification of 40 copies/mL. Patients with a plasma HIV-1 RNA level ≥ 50 copies/mL were counselled on treatment adherence, and had blood samples collected for re-testing at the central laboratory at up to 8-week intervals until the plasma HIV-1 RNA was < 50 copies/mL or the plasma HIV-1 RNA level was confirmed by two consecutive tests to be ≥ 400 copies/mL. Patients with a confirmed plasma HIV-1 RNA level ≥ 400 copies/mL measured at the central laboratory were classified as virologic failures. The confirmatory viral load sample was tested for genotypic drug resistance at the central laboratory.Blood samples were collected at screening, baseline, weeks 4, 12, 24, 36 and 48, and every 24 weeks up to study end or until discontinuation and then at post-treatment follow-up. HIV-1 RNA was measured at a central laboratory, using the Abbott RealTiCD4+ cell counts were determined at a central laboratory at screening, baseline, every 24 weeks up to study end or until discontinuation and then at post-treatment follow-up.Safety monitoring  was performed throughout the treatment phase until study end. Electrocardiograms were recorded at screening, weeks 24 and 48, or at treatment discontinuation if earlier. The following AE classes of interest were investigated based on previous data from the RPV pivotal studies: rashes, neuropsychiatric events, potential QT prolongation-related events, hepatic events and endocrinological events. In addition, hyperglycaemia and new onset diabetes were analysed based on reported AEs during the study.31 Patients were classified as virologic responders if their HIV-1 RNA was < 400 copies/mL within the time window of the week 48 visit (between week 42 and week 58), or if a single HIV-1 RNA value ≥ 400 copies/mL within the time window was not confirmed by a second measurement – the definition of virologic suppression selected was < 400 copies/mL, to reflect the real-life practice in LMICs where a viral load of < 1000 copies/mL should be taken as evidence as suppression.37 Patients with no HIV-1 RNA measurement within the time window of the week 48 visit were considered non-responders.The primary objective was to demonstrate non-inferiority of a TDF/FTC/RPV STR versus TDF/FTC/EFV STR in the percentage of patients with plasma HIV-1 RNA levels < 400 copies/mL after 48 weeks (non-inferiority margin of 10%) using a modified Food and Drug Administration (FDA) Snapshot method.Secondary endpoints were non-inferiority in the percentage of patients with plasma HIV-1 RNA levels < 50 copies/mL after 48 weeks (modified FDA Snapshot method), rates of virologic failure during the 48 weeks of treatment with HIV-1 RNA levels ≥ 400 or ≥ 50 copies/mL , change in CD4+ cell count, loss of treatment options, as defined by treatment-emergent drug resistance, and adherence to study treatment based on tablet count at each study visit up to week 48.Assuming response rates of 90% at 48 weeks for both treatment arms, 192 patients were required per arm to establish non-inferiority of TDF/FTC/RPV versus TDF/FTC/EFV, with a maximum allowable difference of 10%, a one-sided significance level of 2.5%, and 90% power. To account for a maximum of up to 10% major protocol deviations that would result in exclusion of patients from the per protocol (PP) analysis, 213 patients were planned to be recruited in each treatment arm, resulting in 426 randomised patients in total.The primary efficacy analysis was conducted on the intent-to-treat (ITT) population . This analysis was repeated for the PP population (a subset of the ITT population that excluded patients with major protocol deviations). As pre-specified in the statistical analysis plan (SAP), treatment arms were compared using the Cochran–Mantel–Haenszel method, adjusted for the stratification variable (use of EFV vs. NVP at the screening visit). TDF/FTC/RPV was considered non-inferior to TDF/FTC/EFV if the lower limit of the 95% CI of the difference in efficacy was ≥ 10%. Analysis of the percentages of patients with HIV-1 RNA levels < 50 copies/mL, a secondary efficacy outcome, used the same statistical methods as the primary analysis.Subgroup analyses of the virologic response were performed in the ITT population for the following pre-defined groups: NNRTI taken at screening (as stratified), baseline CD4+ count category, sex, country and treatment adherence. The ITT population was used for all safety analyses; as pre-specified in the SAP, there was no formal statistical testing of safety parameters in the study.Ethics committee approval was obtained at all participating centres in accordance with the principles of the 2008 Declaration of Helsinki.Patients were recruited between 23 August 2013 and 14 August 2014. Treatment duration was between 48 and 108 weeks. Of 492 patients screened, 66 were excluded and 426 were randomised (213 in each arm); two patients in the TDF/FTC/EFV arm did not start randomised therapy (one was randomised in error and one withdrew consent). The ITT population comprised 424 patients .Overall, demographic and baseline disease characteristics were well balanced between the two treatment arms . Most paAll patients were taking EFV- or NVP-based regimen at screening. Most patients (415/424 [97.9%]) were taking a non-STR before being enrolled in the study. After randomisation, in the TDF/FTC/EFV arm, 116/211 (55.0%) patients remained on EFV and 95/211 (45.0%) patients changed their NVP for EFV. In the TDF/FTC/RPV arm, 115/213 (54.0%) patients were taking an EFV-based regimen and 98/213 (46.0%) patients were taking an NVP-based regimen at screening. In the TDF/FTC/RPV arm, all (213 [100.0%]) patients changed their NNRTI to RPV at randomisation. In addition, all but two patients (99.1%) had a switch in both their NNRTI and NRTI, while in the TDF/FTC/EFV arm, only 45.5% had to switch both their NNRTI and NRTI. In total, 397 (93.6%) of 424 patients stayed on study medication until study end. Of the patients who discontinued prematurely, 17/213 (8.0%) had received RPV and 10/211 (4.7%) had received EFV. High adherence rates (more than 95% adherence based on tablet count) were documented in 95.8% (204/213) switched to TDF/FTC/RPV and in 97.6% (206/211) switched to TDF/FTC/EFV.p = 0.0003)  was reached by 200/213 (93.9%) patients in the TDF/FTC/RPV arm and 203/211 (96.2%) patients in the TDF/FTC/EFV arm with a difference of –2.3% , demonstrating non-inferiority of TDF/FTC/RPV ( 0.0003) . In the  0.0003) . The res 0.0003)  and 3.In the ITT population, 13/213 patients (6.1%) in the TDF/FTC/RPV arm were non-responders at week 48. Among the non-responders, five patients discontinued because of AEs, seven for other reasons and one patient experienced virologic failure. In the TDF/FTC/EFV arm, 8/211 patients (3.8%) were non-responders at week 48: one patient discontinued because of AEs, six for other reasons and one patient experienced virologic failure. Four patients in the TDF/FTC/RPV arm and one patient in the TDF/FTC/EFV arm who discontinued for ‘other reasons’ did so because they did not re-consent to continue the study at another site after their initial site closed because of administrative reasons. In the PP population, 9/207 patients (4.3%) and 7/207 patients (3.4%) were non-responders in the TDF/FTC/RPV and TDF/FTC/EFV group, respectively.3 in the TDF/FTC/RPV group and 6.1 (140.06) cells/mm3 in the TDF/FTC/EFV group.The mean (s.d.) increase in CD4+ cell count from baseline at week 48 was 26.2 (125.14) cells/mmNo resistance-associated mutations of the pre-defined list  were detected in the samples from two patients who had experienced virologic failure, thus there was no loss of treatment options observed in this study.For patients receiving EFV-based regimens at screening, a virologic response of HIV-1 RNA < 400 copies/mL at week 48 was achieved by 114/116 (98.3%) patients who remained on EFV and 107/115 (93.0%) of those who switched to RPV . For patients receiving NVP-based regimens at screening, the corresponding virologic response rate was 93.7% (89/95) in the TDF/FTC/EFV arm and 94.9% (93/98) in the TDF/FTC/RPV arm . The study was not powered to detect non-inferiority between sub groups of the NNRTI at screening. No major differences were observed when stratifying virologic response by sex, baseline CD4+ cell count, adherence or country .Safety data were collected for all patients up until study end. There were no relevant differences in the incidence of AEs between the two arms, except for Division of AIDS (DAIDS) grade 3 or 4 AEs which were more commonly reported in patients receiving TDF/FTC/EFV compared with TDF/FTC/RPV . Regardless of NNRTI at screening, the incidence of treatment-emergent DAIDS grade 3 or 4 AEs was lower in the TDF/FTC/RPV arm compared with TDF/FTC/EFV. For patients who switched from EFV at baseline, the incidence was 15.7% (18/115) in the TDF/FTC/RPV arm versus 24.1% (28/116) in the TDF/FTC/EFV arm; for those patients who switched from NVP at baseline, the incidence was 22.4% (22/98) versus 29.5% (28/95), respectively.The most frequently reported AEs at least possibly related to the study drugs were vertigo (5 [2.3%] in the TDF/FTC/RPV arm vs. 11 [5.2%] in the TDF/FTC/EFV arm), headache (10 [4.7%] vs. 6 [2.8%]), dizziness (2 [0.9%] vs. 8 [3.8%]), increased appetite (8 [3.8%] vs. 1 [0.5%]) and nightmares (0 vs. 8 [3.8%]) .More patients in the TDF/FTC/EFV arm than in the TDF/FTC/RPV arm experienced grade 3 or higher lipid abnormalities .Adverse events leading to discontinuation were reported in seven (3.3%) patients switched to TDF/FTC/RPV and in one (0.5%) patient receiving TDF/FTC/EFV. All AEs leading to permanent discontinuation were observed in at most one patient in any treatment arm, except for alanine aminotransferase increases and ECG QT prolongation, which both occurred in two (0.9%) patients in the TDF/FTC/RPV arm.In the TDF/FTC/EFV group, a lower rate of treatment-emergent neuropsychiatric events of interest was seen in the subgroup of patients who were receiving EFV at screening  compared with the rate in patients receiving NVP at screening . This difference between the subgroups within the TDF/FTC/EFV group was seen consistently for most individual neuropsychiatric events recorded.In the subgroup switched from NVP-based regimens, 41.1% (39/95) of patients in the TDF/FTC/EFV arm experienced a neuropsychiatric event of interest, compared with only 30.6% (30/98) of the patients who switched to the TDF/FTC/RPV arm .SALIF examined the effect of switching to TDF/FTC/RPV in patients from LMICs with suppressed viral loads who were on an NNRTI-based first-line ART. This study is important because it provides additional data on the utility of TDF/FTC/RPV as a viable alternative for virologically suppressed patients on first-line NNRTI-based regimen in LMICs and in a study population that is predominantly female because women comprised > 60% of the patients enrolled.22 and the NEAR-RWANDA study, which demonstrated non-inferior efficacy and comparable safety of a TDF/FTC/RPV STR versus NVP-based ART in Rwanda.25 Taken together, these data support the use of RPV-based STR regimens in virologically suppressed patients. Given that viral load measurements prior to ART initiation are not routinely conducted in many LMICs, RPV-based STR regimens are an appropriate switch option for patients with suppressed viral loads. These patients have demonstrated sufficient adherence and might benefit from a switch, particularly given concerns around the safety profile of EFV- and NVP-based regimens.The SALIF data add to the evidence from the SPIRIT study, which examined TDF/FTC/RPV STR as a switch option from a PI-based regimen in mostly Caucasian men in high-income settings,42 In addition, the introduction of STRs containing tenofovir alafenamide (TAF) 25 mg instead of TDF 300 mg, or an STR of DTG/RPV, could potentially offer increased long-term tolerability at lower dosing and, eventually, lower costs.45 For virologically suppressed patients such as those in the SALIF trial, who are stable on ART and have already demonstrated high adherence, the risk of virologic failure is considered low. Therefore, switching to an STR may further motivate patients to stay on therapy while leading to fewer medication errors and supporting long-term adherence. This is in line with current normative guidance for mature ART programmes, which recommends differentiated models of care for patients who are stable on ART.46Recent reports from Europe have provided encouraging data on the tolerability of TDF/FTC/RPV in clinical practice, which may be transferable to the LMIC settings.47 therefore, patients in SALIF were closely monitored for these AEs. In studies in treatment-naive patients, most rash events (3% with RPV vs. 14% with EFV) occurred during the first 48 weeks of treatment,21 with few additional patients experiencing rashes during the second year.47 SALIF included patients who had already been successfully treated with NNRTIs, and rashes were rarely seen. EFV can cause neuropsychiatric side effects, which often resolve within the first weeks of treatment.10 This study could not confirm the improved central nervous system tolerability of RPV compared with EFV seen in treatment-naive patients.21 However, the subgroup analysis of patients who switched from NVP to either RPV or EFV showed tolerability differences in favour of RPV.Increased rates of rash and neuropsychiatric events have been reported following RPV and EFV treatment;48 This suggests that our safety findings are subject to some inherent bias of the study design. This negative bias might explain why the ITT analysis showed no differences in tolerability, while other studies in treatment-naive patients have demonstrated a generally more favourable tolerability profile of RPV compared with EFV.47 A control group of non-switchers (or deferred switchers) staying on their original ART might help address such inherent biases in future switch studies. Another limitation of the study is the open-label design, which may influence the reporting of side effects and discontinuation rates. For instance, QT prolongations were reported in three patients on TDF/FTC/RPV and three patients receiving TDF/FTC/EFV; while two of the three patients receiving TDF/FTC/RPV discontinued their regimen, none in the EFV arm discontinued. A caveat to the generalisability of the study results is that trial candidates with CD4 cell counts < 200 cells/mm3 were excluded. Also, it should be noted that the definition of viral suppression used in the study was < 400 copies/mL rather than < 50 copies/mL; this was chosen to reflect real-life practice in LMICs and to account for blips, and is within the recommended WHO guidance to use a definition < 1000 copies/mL in LMICs. Finally, our study required participants, as an inclusion criterion, to have access to at least one meal a day, a situation that does not necessarily always pertain in sub-Saharan Africa and other LMIC regions.The strength of this study is that it illustrates the benefit of STRs for patients in resource-limited settings who have tolerability issues with currently available NNRTIs and who are already virologically suppressed. The main limitation of the study was that all patients entering the TDF/FTC/RPV arm changed the previous NNRTI component of their regimen, while > 50% of the patients in the TDF/FTC/EFV arm had previously received an EFV-based regimen. Switching to a new regimen may confer a potential risk for new tolerability or safety issues. Furthermore, the treatment-emergent neuropsychiatric events in the subgroup of patients switching from EFV are discordant with general tolerability data .In adults from LMICs with suppressed viral load on first-line NNRTI-based therapy, switching to an STR of TDF/FTC/RPV was non-inferior to an STR of TDF/FTC/EFV in maintaining high rates of viral suppression, with comparable safety at 48 weeks. Our findings support the use of TDF/FTC/RPV as a viable alternative to both EFV- and NVP-based regimens in LMICs, where access to a wider variety of affordable ART options is urgently needed.


In resource-limited settings holding regimens, such as lamivudine monotherapy (LM), are used to manage HIV-positive children failing combination antiretroviral therapy (cART) to mitigate the risk of drug resistance developing, whilst adherence barriers are addressed or when access to second- or third-line regimens is restricted. We aimed to investigate characteristics of children placed on LM and their outcomes.We describe the characteristics of children (age <16 years at cART start) from 5 IeDEA-SA cohorts with a record of LM during their treatment history. Among those on LM for >90 days we describe their immunologic outcomes on LM and their immunologic and virologic outcomes after resuming cART.We included 228 children in our study. At LM start their median age was 12.0 years (IQR 7.3–14.6), duration on cART was 3.6 years (IQR 2.0–5.9) and median CD4 count was 605.5 cells/μL (IQR 427–901). Whilst 110 (48%) had no prior protease inhibitor (PI)-exposure, of the 69 with recorded PI-exposure, 9 (13%) patients had documented resistance to all PIs. After 6 months on LM, 70% (94/135) experienced a drop in CD4, with a predicted average CD4 decline of 46.5 cells/μL (95% CI 37.7–55.4). Whilst on LM, 46% experienced a drop in CD4 to <500 cells/μL, 18 (8%) experienced WHO stage 3 or 4 events, and 3 children died. On resumption of cART the average gain in CD4 was 15.65 cells/uL per month and 66.6% (95% CI 59.3–73.7) achieved viral suppression  at 6 months after resuming cART.Most patients experienced immune decline on LM. Its use should be avoided in those with low CD4 counts, but restricted use may be necessary when treatment options are limited. Managing children with virologic failure will continue to be challenging until more treatment options and better adherence strategies are available. In resou.–5 Switch.Lamivudine monotherapy (LM) is one such holding regimen. This commonly used holding strategy reduces the pill burden to patients and allows time for adherence barriers to be addressed. It is also used whilst awaiting access to second- or third-line regimens, not always readily available. Lamivudine has a low genetic barrier to resistance and most patients with virologic failure develop the M184V mutation. LM retaiThere are few studies of the use and effectiveness of LM in children, despite it being recommended in national guidelines. Three small South African studies found that CD4 count declined slowly on LM and those with higher CD4s did not experience significant declines.–17 We aiData were collected prospectively from five IeDEA-SA cohorts in South Africa. IeDEA is a multi-regional HIV cohort collaboration, which has been previously described. The requWe included data from children (<16 years) initiating cART after 2004, with a record of being on LM at some point in their treatment history. Outcomes and more detailed analysis was performed on patients who received LM for more than 90 days. Database closure ranged from October 2015 to February 2016 across sites.All sites made use of routine viral load (VL) monitoring and are part of the South African national ART program. The following treatment guidelines were in place during the study period: Before 2007 the recommended first-line regimen was stavudine, lamivudine and either efavirenz, or, for those starting treatment younger than three years of age, lopinavir/ritonavir (LPV/r). For those with tuberculosis or starting treatment under the age of six months, LPV/r was replaced with ritonavir alone. After 20The recommended second-line regimen prior to 2010 was to change at least one of the nucleoside reverse transcriptase inhibitors (NRTIs) and replace efavirenz with LPV/r (if on efavirenz-based first-line), or switch to non-nucleoside reverse transcriptase inhibitor (NNRTI)-based second-line for those on protease-inhibitor (PI) based first-line. The recoDemographic, laboratory and clinical data at cART start and at the start of LM included age, gender, CD4 count and CD4%, age-adjusted weight and height z-scores, WHO stage, immune status according to WHO 2006 criteria for immunosuppression, and firsa priori. For models relating to characteristics after cART resumption after LM we included age at LM start, current regimen after resuming cART, duration on LM, treatment interruption prior to LM, nadir CD4, CD4 at cART resumption, age at cART resumption, exposure to PI-based cART prior to LM. Final models were built by sequentially excluding variables through backwards selection using p-values. Statistical analysis was performed using Stata version 15 .In this analysis, we described the characteristics of patients receiving LM summarizing continuous and categorical variables using medians and interquartile ranges, and proportions respectively. For patients with multiple episodes of LM, the first episode of at least 90 days was used. To better understand immunologic outcomes we explored mean changes in CD4 count whilst on LM, as well as immune decline, which we defined as either a drop in CD4 below 500 cells/μL for those starting LM with CD4 above 500 cells/μL, or for those starting LM with CD4 count of 500 or below, a drop in CD4 of more than 10%. We used Kaplan-Meier estimates for children on LM for at least 90 days and with at least 6 months of follow-up on LM to predict probability of immune decline whilst on LM. Survival was calculated from the date of LM start until either the date that immune decline was recorded, death, or censoring at the last clinical visit recorded before database closure. Predictors of immune decline whilst on LM were determined using Cox-proportional hazards models adjusting for age at cART start, characteristics prior to LM start including treatment interruption, nadir CD4 count and type of cART regimen, as well as duration on LM. To account for the non-independence of repeated CD4 measures taken for each patient, we used a linear mixed-effects regression models to determine the mean change in absolute CD4 count at 6 months on LM for patients with different characteristics at LM start. We also explored the immunologic and virologic outcomes for patients who resumed cART and had at least one subsequent CD4 count or VL. Kaplan-Meier estimates were used to predict the probability of virologic suppression (VL<1000) for those who resumed cART after LM. We used Cox-proportional hazards models to determine predictors of virologic suppression after cART resumption adjusting for age at LM start and current regimen after resuming cART. To explore the immunologic changes after cART resumption we calculated the CD4 slope for each child, as the difference between the CD4 at resumption of cART (up to 90 days prior and 7 days post cART resumption) and the first CD4 count taken within a year of cART resumption, divided by the time in months between these two measurements. We used linear regression to explore associations with change in CD4 after cART resumption adjusting for CD4 at cART resumption (> = 500 or <500), age at cART resumption and exposure to PI-based cART prior to LM. For multivariate models we included a number of variables relating to characteristics at cART start , at LM start  and whilst on LM  In total, 228 patients  with a history of being on LM were included in the study. At cART start, median age was 7.4 years (IQR 3.2–10.0), median CD4 count was 348 cells/μL (IQR 190–616), median CD4% was 12.7% (IQR 7.6–18.0), and 118 (52%) were immune-suppressed. Most patients were initiated on efavirenz-based first-line regimens (70%), with 57 (25%) on PI-based first-line .10 (IQR 3.7–4.7). Median time with unsuppressed VL, calculated as the total time between the first consecutive unsuppressed VL measurement and the date of LM start, was 421.5 days (IQR 234.5–794), with a median of 4 (IQR 2–5) unsuppressed VL measurements. Time with unsuppressed VL prior to LM has decreased in more recent years: among those with at least 2 years of follow-up following their first consecutive unsuppressed VL and those starting LM within 2 years of their first consecutive unsuppressed VL, the median time with unsuppressed VL declined (2002–2005: 371 days (IQR 259–519), 2006–2009: 285 days (IQR 151–472), 2010–2014: 280 days (IQR 201–425)). Prior to LM start, 110 (48%) were on efavirenz-based first-line regimens, whilst 108 (47%) had had prior exposure to PI-based regimens, one patient was on a third-line regimen, two had been on other holding regimens, and seven had unknown treatment history.At LM start the median age was 12.0 years (IQR 7.3–14.6) and patients had been on cART for a median of 3.6 years (IQR 2.0–5.9), with median CD4 and CD4% of 605.5 cells/μL (IQR 427–901) and 22.2% (16.2–28.2), respectively; median VL was 4.2 logPrior to initiating LM, 116 (51%) had undergone resistance testing. Among those tested, 107 (92%) patients had resistance to lamivudine with the majority having high level resistance . Over half of patients had no PI resistance , whilst 10 (9%) were resistant to all PI’s.Of 69 patients with recorded PI-exposure prior to LM and a resistance test, 38 (55%) had no PI resistance, 10 (15%) were resistant to only nelfinavir, 12 (17%) were resistant to some PIs and 9 (13%) were resistant to all PIs.At study closure the median duration on LM was 309 days (IQR 94.5–644), and 174 (76%) children were on LM for more than 90 days. Four patients had been on LM on multiple occasions between periods of receiving cART.Three patients died whilst on LM, although details on cause of death were not available. All had a CD4 at LM start >500 cells/μL and had previously been on PI-based cART . Time on LM for the 3 patients ranged from less than 6 months to over 18 months, one patient dropped to a CD4<50 whilst on LM. In total, there were 114 new disease events among 53 patients whilst on LM. The median time to first disease event was 94.5 days (IQR 45–359). There were 18 (8%) patients who experienced 26 WHO stage 3 or 4 event whilst on LM, the most common being extra pulmonary tuberculosis (5), recurrent bacterial pneumonia (4), oral candidiasis (3), chronic HIV-associated lung disease (3), and acute necrotising ulcerative stomatitis, gingivitis or periodontitis (3).At study closure, out of all 228 children, 167 patients had been switched from LM, 149 of whom resumed cART. The majority, 135, were switched to or resumed PI-based regimens , and 12 switched to or resumed NNRTI-based regimens . One patient was switched to a third-line regimen, after having been on PI-based second-line prior to LM, and one patient was switched to a non-standard cART regimen. Among the 18 patients not resuming cART, 4 were switched to other holding regimens , and 14 discontinued all treatment. We did not have data on the reasons for treatment discontinuation, and the decision to stop all treatment may have been planned and recommended by a clinician, or the decision of the patient.In total 147 patients were on LM for more than 90 days, had a recorded CD4 count at LM start, and at least one subsequent CD4 count measured whilst receiving LM. The median time from the CD4 count at LM start to the last CD4 on LM was 413 days (IQR 231–752). On average CD4 count measurements were taken every 132 days or every 4.3 months whilst on LM, and the median time between CD4 measurements on LM was 112 days (IQR 91–158). The median CD4 decline, from CD4 at LM start to the last recorded CD4 count on LM, was 148 cells/μL (IQR 28–414), 115 (78%) experienced a drop in CD4 ≥10%, and among the 112 with CD4 ≥500 cells/μL at LM start 46% experienced a drop in CD4 to <500 cells/μL whilst on LM.Results from our mixed effects model predicted an average CD4 decline at 6 months on LM of 46.5 cells/μL (95% CI 37.7–55.4) from a mean CD4 of 708.4 to a mean of 661.9 cells/μL (95% CI 601.8–721.9). CD4 count at LM start influenced immune decline on LM. The predicted average decline in CD4 at 6 months on LM for those with CD4 <500 was 21.5 cells/μL (95% CI 9.7–33.2) to a mean CD4 of 306.8 cells/μL (276.2–337.5). For those with CD4 ≥500 at LM start, the predicted decline at 6 months was 55.5 cells/μL (95%CI 47.4–63.6), to a mean CD4 of 752.3 cells/μL (95% CI 704.4–800.2). To illustrate the difference in CD4 decline for patients of different characteristics, we estimated the average CD4 count at 6 months on LM for patient of varying age, duration on cART, nadir CD4 and CD4 at LM start. These results are summarized in According to our definition, 76 (52%) patients experienced immune decline on LM, with an estimated probability of immune decline after 6 months on LM of 22.2% (95% CI 16.6%-29.3%). Predictors of immune decline are summarized in Among the 103 patients resuming cART after at least 90 days on LM, 62% (64) achieved virologic suppression after resuming cART, with estimated probability of VL suppression by 6 months after cART resumption of 66.6% (95% CI 59.3–73.7). In adjusted analysis, those on PI-based cART were more likely to suppress compared to those resuming NNRTI-based cART . Age was associated with VL suppression, with those 5 years or younger (aOR 2.1 (95% CI 1.6–2.7), those 5 to 10 years of age (aOR 1.9 (95% CI 1.5–2.3) and those aged 10 to 13 years (aOR 1.1 (95% CI 0.8–1.3) more likely to suppress compare to those older than 13 years. Regimen prior to LM was not associated with virologic suppression after resumption of cART and was not included in the final model.Among patients on PI-based regimens prior to LM, on resumption of PI-based cART 21/36 (58%) achieved VL suppression. Among the 46 patients on NNRTI-based first-line prior to LM, a third (2/6) patients suppressed on NNRTI-based regimens after resuming cART, while 31/40 (78%) suppressed on PI-based cART.The majority of children placed on LM experienced declines in CD4, 8% experienced WHO stage 3 or 4 events, and 3 children died. Those with higher CD4 count at LM start, lower nadir CD4 and older age experienced bigger absolute declines in CD4 count. Our composite outcome of immune decline  was experienced by roughly half of patients. Predictors of immune decline were lower nadir CD4 count, and having a history of treatment interruption. After resuming cART, the majority of patients achieved virologic suppression and some immune recovery. Adolescents and those on NNRTI-based cART were less likely to achieve virologic suppression on resumption of cART.Most children placed on LM had failed to suppress virologically for over a year. Time with unsuppressed VL prior to LM has decreased in more recent years suggesting a trend to earlier adoption of this strategy. About half our population had limited treatment options and were placed on LM after exposure to PI-based regimens, with 10 children having documented resistance to all PIs. The other half of children placed on LM had only had exposure to NNRTI-based first-line regimens and were placed on this strategy instead of switching directly to PI-based second-line. After LM few of these children resumed NNRTI-based cART and very few managed to suppress on this regimen. The majority resumed PI-based regimens and had good virologic outcomes. For these children being on LM delayed the initiation of second-line whilst risking further immune decline, although it is possible that barriers to adherence were addressed during this time so that adherence was optimized by the time second-line was started.This study represents the largest cohort of children on LM, providing the most evidence on the use of this strategy. We explored outcomes of patients on resumption of cART following LM; only one US-based study of 36 patients has reported on similar outcomes. Like allWe had no data on adherence, and therefore could not directly assess the effect of LM as a strategy to improve adherence, or determine how much adherence influenced immunologic outcomes on LM or outcomes on resumption of cART. We did not have data on the reason for starting the patient on LM, and could only infer from the patient’s treatment history. Resistance testing was not available for all patients. VL monitoring is not routinely performed for those on non-supressive regimens like LM, so we could not assess the virologic consequences of this strategy.Our study had a larger and older study population than those previously published, but our results are largely in agreement. Linder et al reported generally larger immune declines, but children in their study started LM with lower CD4 counts, and had longer durations on LM (mean duration of over 2 years compared to our median of under 1 year). A similaThere has been some attempt to prospectively compare outcomes of holding regimens like LM, with other strategies to manage virologic failure in children. The IMPAACT P1094 study enrolled children failing non-NNRTI-based cART, and aimed to compare outcomes of those randomized to continue with failing cART versus changing to a holding regimen of lamivudine or emtricitabine monotherapy. The study was discontinued due to lack of enrolment, but results from the small study population indicate greater immune decline for those on holding regimens. Fairlie This study provides useful evidence on the outcomes of children on LM and after resuming cART, providing guidance on which patients are at greater risk of immune deterioration on LM. This strategy should be avoided among children with low CD4 counts, and patients on LM require careful monitoring. Half of our study population were failing PI-based regimens and had limited treatment options available to them. As more HIV-positive children are started on PI-based cART due to earlier HIV diagnosis, it is likely that an increasing number of children will experience virological failure on PI-based cART. As long as treatment options for children remain scarce, and the risk of poor adherence and resistance continues, the use of holding regimens like LM, could increase as the numbers of children failing treatment grow. There is an urgent need for evidence to inform the optimal management of children with virological failure, as well as to inform second- and third-line options for children in resource-limited settings. As new aTo understand how best to manage virologic failure in children and adolescents, comparison between switching patients directly to second-line, keeping them on their failing regimen or discontinuing all treatment is needed. This will allow a better understanding of the benefits and potential risks of using holding regimens like LM.In conclusion, most patients experienced immune decline on LM, and its use should be avoided in those with low CD4 count. LM may be a useful strategy in the management of patients with virologic failure and poor adherence when treatment options are limited, but close clinical and immunological monitoring is required. Managing children with virologic failure will continue to be challenging until more treatment options and better adherence strategies are available.


Plasmodium falciparum with some, but not all, of the reported antifolate resistance mutations reported in Uganda. With TMP–SMX prophylaxis, suboptimal adherence is concerning. Sulfamethoxazole levels below IC50s required to overcome malaria parasites with multiple antifolate resistance mutations may be significant. Further study of TMP–SMX in this context is needed.Trimethoprim–sulfamethoxazole (TMP–SMX) prophylaxis in HIV-uninfected, exposed (HUE) children variably reduces clinical malaria burden despite antifolate resistance, but data regarding achieved serum levels and adherence are lacking. Serum samples from 70 HUE children aged 3–12 months from Rakai, Uganda, enrolled in an observational study were assayed for random SMX levels using a colorimetric assay. Adherence with TMP–SMX prophylaxis data (yes/no) was also collected. Of 148 visits with concurrent SMX levels available, 56% had self-reported adherence with TMP–SMX therapy. Among these 82 visits, mean (standard deviation) level was 19.78 (19.22) µg/mL, but 33% had SMX levels below half maximal inhibitory concentrations (IC50) for  Studies have shown that when HIV and malaria are present as coinfections, each disease can enhance the pathogenicity of the other.2 Many HIV-uninfected, exposed (HUE) children in sub-Saharan Africa reside in malaria-endemic areas. Trimethoprim–sulfamethoxazole has been shown to have varying degrees of antimalarial impact even in the face of high antifolate resistance prevalence,4 but few studies have examined that TMP–SMX achieved levels and adherence in these populations. Herein, we report SMX levels (as a component of TMP–SMX) and adherence in a population of HUE children in an observational study.The World Health Organization recommends daily trimethoprim–sulfamethoxazole (TMP–SMX) prophylaxis for children of HIV-infected mothers daily starting at 4–6 weeks of age and continued until HIV infection has been excluded by an age-appropriate HIV test, after cessation of breastfeeding.5 Children of HIV-infected mothers were breastfed, as per World Health Organization (WHO) recommendations, up to at least 12 months of life,5 and HIV tests were performed after birth and once after the cessation of breastfeeding (HIV exposure) at Kalisizo hospital as per standard of care.5 Mothers of HUE children also received standard of care “Option B+,” which entails treating the mother with triple antiretroviral therapy (ARV) as soon as she is diagnosed and continuing for life, regardless of CD4 count. Triple ARV treatment refers to the use of one of the recommended three-drug fully suppressive treatment options, including AZT, NVP, or lamivudine.6Infants aged 0–12 months from Rakai district were eligible for enrollment and followed up at the Kalisizo Hospital and Rakai Health Services Program. Subjects were followed up monthly from February 2015 to August 2015. The original study design included enrollment of HUE and HIV-uninfected, unexposed children (HUU) with a primary objective of characterizing malaria incidence, but was stopped early for futility because of low malaria incidence in the region. This report therefore presents data from an exploratory objective. No drug was administered as part of this observational study, but children received standard-of-care management in Uganda, which for children of HIV-infected mothers involves daily nevirapine (NVP) or azidothymidine (AZT) from birth through 4–6 weeks of age, regardless of infant feeding method, along with daily prophylactic doses of TMP–SMX starting by 6 weeks of age and continuing until 6 weeks after breastfeeding is discontinued.At study visits, history and physicals were performed, TMP–SMX adherence data (yes/no) were collected , and heel/finger stick and venous blood collected for drug levels (SMX of TMP–SMX). To preserve sample stability, the samples were stored at −80°C immediately after collection and processing on site in Uganda. The samples were shipped back on dry ice (with temperature monitoring and no thawing). Once received, the samples were immediately stored at −80°C until use for the assay. If any samples needed to be rerun, that sample was kept at −20°C after the initial run and until use for the next run.7 and WHO malaria treatment recommendations.8 Dried blood spots were also collected for malaria polymerase chain reaction, performed as previously described.9 Maternal HIV status was determined from documented medical history. All subjects received insecticide-treated bednets to prevent malaria if they did not already have them.At all visits, children were tested for malaria using Giemsa-stained malaria thick smear. Clinical illness was managed according to Integrated Management of Childhood Illness guidelines10 Malaria is meso- to holoendemic with year-round transmission and highest intensity after the rainy seasons or in communities adjacent to lakes and other mosquito breeding sites.11Rakai district registers an HIV seroprevalence of 8.5% among the pregnant mothers across the various geographical populations, with an estimated 2,000 births per year . Rakai district is on a plateau at an altitude ranging between 750 and 900 m and has fair rainfall throughout the year, with relatively dry periods during January and February and from June through August. Peak rainfall varies from year to year, but occurs typically in March/April and October/November.12 with some modifications, including adaptation to 96-well plates. Briefly, 20 μL of serum was diluted in 260 μL of water, incubated at room temperature for 5 minutes, and boiled (98°C) for 1 hour in a thermocycler to deacetylate the small portion of SMX, which is naturally acetylated in the body. The plate was then centrifuged at 2,000 rpm for 3 minutes. A 120-μL quantity of 20% p-toluenesulfonic acid in 0.2 M HCl was added to each sample and incubated for 5 minutes to precipitate serum proteins. The plate was then centrifuged at 4,000 rpm for 10 minutes. A 100-μL quantity of supernatant was recovered and combined with 20 μL of a citric acid buffer, followed by 40 μL of a 2% dimethylaminobenzaldehyde solution in ethanol, resulting in a color change that was quantified at 450 nm as a measure of sulfa levels. The assay was standardized using control serum, to which SMX of known level was added in serial dilutions. Our limit of detection was 1.9 μg/mL and, therefore, levels equal to 1.9 μg/mL or less were considered undetectable. Standard curves were included with each plate to allow for determination of sample level. Samples with values above 5.8 μg/mL were considered therapeutic for IC50 for parasite strains in this regions, based on in vitro growth inhibition assay IC50 data for SMX with Plasmodium falciparum strains with known antifolate resistance mutations13 and known resistance mutations of F32 and K1 compared with resistance data published for Uganda, indicating a range of 2.53–5.8 μg/mL.15Sulfa levels in serum were measured using a previously described colorimetric assayR version 3.3.1 was used for all calculations, including summary statistics .The study was approved by the Uganda Virus Research Institute Research and Ethics Committee, the Uganda National Council for Science and Technology, and the National Institute of Allergy and Infectious Diseases Intramural Institutional Review Board. Parents or guardians of infants and children enrolled provided written consent for the infants enrolled in this study.Supplemental Table 1.Seventy HUE subjects were enrolled, aged 3–12 months while on study with a mean duration on study of 11.78 weeks (SD 6.84). Mean age at enrollment was 6.76 (2.37) years . Demogra15)  µg/mL. As well, 33% of concurrent drug levels were below 5.8 μg/mL (IC50 range 2.53–5.8 μg/mL15) .P. falciparum with some, but not all, of the already reported antifolate resistance mutations that exist in Uganda . However, with all those caveats taken into account, it is still concerning to note that if we further adjusted our random SMX levels to account for the amount of protein-bound (70%) as opposed to free (active) drug (30%), levels are considerably lower than IC50s.20Weaknesses of our study include that we were not able to perform more detailed pharmacokinetics of TMP–SMX on these children. In addition, we acknowledge certain caveats in attempting to interpret available in vitro Although TMP–SMX was not originally intended to treat or provide prophylaxis against malaria, the HIV pandemic in areas of malaria endemicity presents continued questions surrounding TMP–SMX impact on malaria. Further studies are required.Supplemental Table.