HIV management remains concerning and even more challenging in the frame of comorbidities like malnutrition that favors disease progression and mortality in resource-limited settings (RLS).To describe the nutritional parameters of antiretroviral therapy (ART) recipients  with respect to CD4 count and virological failure.A cross-sectional study was conducted from October to December 2018 among 146 consenting participants enrolled in two health facilities of the East-Region of Cameroon. Socio-demographic data, basic clinical information and treatment history were collected; blood samples were collected by venipuncture for laboratory analysis  performed at the “Chantal Biya” International Reference Center”, Yaounde, Cameroon. The nutritional profile was assessed by using anthropometric and biochemical parameters. Data were analyzed using Excel 2016, Graph pad prism version 6; Spearman correlation and Kruskal-Wallis test were used; with p<0.05 considered statistically significant.Median [IQR] age was 42 [33–51] years, 76.0% (111/146) were female and median [IQR] duration on ART was 54 [28–86] months. Of these participants, 11.6% (17/146) were underweight based on the body mass index and 4.7% (7/146) were at the stage of advanced weight loss. According to immunovirological responses, 44.5% (65/146) were immunocompromised (CD4<500 cell/μl) and 75.3% (110/146) had an undetectable viremia (<40 copies/mL). CD4 count inversely correlated with total protein concentration . Viremia was inversely correlated with albumin , nutritional risk index , total cholesterol , and positively correlated with total protein  concentrations.In this RLS, with patients having about five years of ART-experience, malnutrition appears to be driven mainly by a poor BMI, indicating that about one of ten patients falls within this severe condition. However, the largely normal nutritional profiles should be interpreted with caution, considering local realities and food support programs in place. The present outcomes highlight the need for monitoring nutritional status of people receiving ART in RLS, toward the design of optimal food interventions. The human immunodeficiency virus (HIV) targets the immune system and weakens the surveillance by the body’s own defense system against cancer cells and infections, which in turns leads to susceptibility of HIV infected individuals to a wide range of infections normally cleared by the immune system of a healthy/immunocompetent individual . HIV canMalnutrition is one of the major complications of HIV infection  and has HIV/AIDS and malnutrition seemingly have a synergistic interaction within the host, characterized by interaction between a poor nutritional status and HIV infection (driven by progressive degradation of immune system). This leads to the development of opportunistic infections and increases the risks of malignancies, CD4 T-cell depletion and death , 11. In Progress in scaling-up HIV treatment  have reduced associated mortality and morbidity, thus making HIV a chronic infection, even in sub-Saharan African (SSA) countries where 70% of the global epidemics is concentrated –18. ThusIn spite of the declining burden of HIV at country-level (5.4% in 2004 to 2.7% in 2019), Cameroon is still experiencing a generalized epidemiology. The national strategy for the fight against HIV/AIDS in Cameroon recommends a nutritional supplement to subside the current 14.1% persistent rate of malnutrition faced by PLHIV, which intends to support ART response , 23. IntWe therefore sought to describe the nutritional parameters of ART recipients in real-life  with respect to CD4 count and virological failure in the East-region of Cameroon.A prospective, cross-sectional and analytical study was carried-out among ART-experienced HIV-infected patients at two heath facilities in the East region of Cameroon, Bertoua Regional Hospital (BRH); and Nkolbikon Catholic Health Center (NCHC), during three months (from October through December 2018).A standard questionnaire  was admiFollowing a convenience sampling strategy, participants were enrolled consecutively based on the following inclusion criteria: (1) having a confirmed HIV-positive result; (2) be on ART for at least six months; (3) be registered in one of the study sites; and (4) aged 15 years and above. Those excluded from our study were: (1) patient enrolled in a nutritional program or food-interventional project, or (2) those having incomplete data related to any of the study parameters. It is worth noting that excluding patients already in a nutritional program helps in limiting selection bias and in ensuring generalizability of the findings to the target population.Blood sample was collected (in dry and ethylene diamine tetra-acetic acid test tubes of 4 ml) by venipuncture with the help of trained phlebotomists. After collection, blood samples were transferred from the sampling sites to the BRH laboratory for packaging and shipment. Only dry test tubes were centrifuged and separated in aliquots for biochemical analysis. After centrifugation, racks of all samples were placed in ice packed isothermal bags, and samples were then sent for laboratory analysis to the study reference institution , located in Yaoundé, the capital city of Cameroon.At the study site, anthropometric parameters were collected from each participant immediately after the interview using a scale (use to collect weight), a measuring board (used to collect height) and the patient’s report .http://www.nsplucknow.com/pdf/CyFlow_Counter_Bro_EN.pdf). CD4 results were then interpreted as follows: no immunodeficiency ; mild immunodeficiency (between 350 and 499); advanced immunodeficiency (between 200 and 349) and severe immunodeficiency (below 200) ;The NRI, a nutritional status assessment index recommended in national nutritional programs, was estimated by the following formula:ght(kg);WLP is used to assess the risk of malnutrition and determined by the formulatweight;According to each of the studied nutritional parameters, the level of malnutrition was defined as severe if the global acute malnutrition (GAM) rate was above 10%, as per the United Nations guidelines .Collected data were entered on Microsoft Excel 2016 sheets and used for basic analysis  The software GraphPad Prism version 6.0. was used for correlation tests. Correlation analysis were performed using Spearman correlation and Kruskal-Wallis test was used to analyze the median (IQR) of nutritional parameters according to categories of CD4  and VL . A statistical significance was considered with p<0.05. Dependent variables were immune status and viremia, while independent variables were anthropometric and biochemical parameters.0 2018/06/1055/CE/CNERSH/SP); written informed consent were obtained from all the participants; data were processed using unique identifiers to ensure confidentiality; laboratory results were returned to participants for possible benefits in their clinical management; and counseling on good nutritional habits and healthy lifestyles was provided to all .The study was conducted in compliance with the core principles of the Helsinki declaration: an administrative authorization was issued; ethical clearance was obtained from the National Ethics Committee for Research on Human Health  at Bertoua regional hospital (BRH) and 11.6% (n = 17) at Nkolbikon Catholic Health Center (NCHC). Among them, 76.0% (n = 111) were females giving a sex ratio  of 3. The median age was 42 [IQR: 33–51] years .The median duration on ART was 54 [IQR: 28–86] months. All participants were on first-line ART regimen consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) and one non-NRTI (NNRTI).Of the 146 participants, BMI results revealed that only a proportion of 11.6% (n = 17) was undernourished. According to NRI, 91.1% (n = 133) were normal, while referring to WLP, 60.9% (n = 89) had a normal weight. Only 2.7% (4) of participants were hypoalbuminemic, 19.9% (n = 29) hyperproteinemic and 20.5% (n = 30) hypocholesterolemic.The median [IQR] CD4+ T cell lymphocyte count was 547 [385–759] cells/μl, and 44.5% (n = 65) were immunocompromised (<500 cells/μl). The median [IQR] of low CD4 (below 200 cell/μl) according to BMI was 21.2  and to WLP was 1.7 [-1.31–4.9]%. The majority (75.3%) of the 146 study participants had a controlled of viral replication (<40 copies/mL) while 12.3% (n = 18) were experiencing virological failure (>1000 copies/mL). The median [IQR] of high viremia (≥1000 copies/mL) according to total cholesterol was 1.5 [1.3–1.6] g/l and to WLP was 1.6 [-1.4–6.2]% .We found a negatively weak correlation between CD4+ T cells count and total protein concentration  as shown on The present study intended to correlate ART response and the nutritional profile among patients receiving ART within the Cameroonian context. Our findings revealed that the majority of our study participants were predominantly female, as reported by previous studies in the country. Similarly, the median age from our study also falls within the age range of the predominant population of PLHIV in Cameroon , 30, 31,Regarding the analysis of anthropometric measurements, about 1 out of 10 participants were undernourished as defined by a poor BMI, suggesting that this poor nutritional status in the Cameroonian context would represent a high public health concern among patients receiving ART. Interestingly, similar findings were recently reported among ART-compliant recipients in Ethiopia; this underscores the fact that beyond adherence to an effective ART-regimen, malnutrition remains a threat that deserves special considerations in the SSA context . FurtherRegarding the analysis of micronutrients, most nutritional parameters were within normal ranges. Of note, the normal total protein profile of the large majority of our population indicates up-regulation of antibody production in an attempt to compensate ongoing immunodeficiency . This isRegarding ART response, the majority of participants achieved viral undetectability and viral suppression similar to previous reports , 43. RegThe present study had some limitations. Of note, the geographical coverage was limited to a single region of Cameroon and the enrollment was non-randomized, which limit somehow the generalizability of our findings. Also, it would have been insightful to assess pro and anti-inflammatory markers with regards to ART response and variability of the nutritional status. It is plausible that the largely normal nutritional profiles found in our study could be due to the fact that patients experiencing malnutrition locally would have been enrolled into the nutrition interventional program onsite, and therefore excluded from our study population . Furthermore, only self-reported adherence has been used to evaluate effects of ART on participants; the cross-sectional design of our study does not give room for causality assessment of HIV on malnutrition, which underscores the relevance of conducting cohort-studies for a deeper understanding of the potential interaction between the two. Moreover, our findings should be cautiously interpreted because of the large number (n = 22) of statistical tests performed .In this RLS, with patients having about five years of ART-experience, malnutrition appears to be driven mainly by a poor BMI, indicating that about one of ten patients falls within this severe condition. However, the largely normal nutritional profiles should be interpreted with caution, considering local realities and food support programs in place. The present outcomes highlight the need for monitoring nutritional status of people receiving ART in RLS, toward the design of optimal food interventions.S1 TableBRH: Bertoua regional hospital; NCHC: Nkolbikon Catholic Health Center, BMI: Body Mass Index; NRI: Nutritional Risk Index; WLP: Weight Loss Percentage.(PDF)Click here for additional data file.S2 TableBMI: Body Mass Index; NRI: Nutritional Risk Index; WLP: Weight Loss Percentage; N: Effective; KWT: Kruskal-Wallis test; P: P Values.(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Antiretroviral treatment (ART) adherence rates are lower among adolescents living with HIV (ALHIV) than among adults and children, but more evidence is needed on long‐term sustained ART adherence among ALHIV. This study assesses rates of sustained ART adherence in a cohort of adolescents in South Africa.A prospective cohort of adolescents (10‐19 years) living with HIV  in the Eastern Cape Province in South Africa were interviewed at baseline (2014‐15) and followed‐up twice . All adolescents ever initiated on treatment in 52 government health facilities were traced  and their clinic records were extracted where available. We investigate sustained ART adherence among adolescents interviewed at all three waves of data collection (N = 933). To quantify adherence at each study wave, we used self‐reported past‐week adherence (including weekdays and weekends). Self‐reported adherence was validated using HIV‐1 RNA viral load (>50 copies/mL cut‐off) reported in clinic records, in a random‐intercept logistic regression.P = 0.007) and adolescents with horizontally acquired HIV (P = 0.002) were more likely to report inconsistent adherence across time. Controlling for socio‐demographic characteristics, past‐week adherence was associated with non‐detectable viral load . Overall, of the adolescents with viral load measurements at study Wave 1 and Wave 2, 50.6% maintained undetectable viral load for the preceding year.At baseline, approximately 66% (N = 615) of adolescents reported past‐week ART adherence, and of these 45.3% reported adherence at both baseline and follow‐up. Only 37.1% of the sample reported sustained past‐week ART adherence over the three waves of the study. Most adolescents  report inconsistent adherence across time (including 6.4% disengaged from care). Older (Adolescents living with HIV reported very low rates of sustained ART adherence. Adherence reported at a single time may mask high rates of variability in adherence over time. These findings highlight the urgent need for enhanced and effective interventions to assist ALHIV with ART adherence through the challenging years of adolescence. Self‐re2This report used data from the Mzantsi Wakho study, a prospective cohort of ALHIV. Adolescents aged (10‐19) years at baseline were recruited from a municipality in Eastern Cape Province in South Africa, a province with an estimated overall HIV prevalence of 14% . ParticiVoluntary informed consent was obtained from adolescents and caregivers when adolescents were <18 years old. Ethical approvals for the Mzantsi Wakho study were granted by the University of Cape Town (UCT/CSSR/2013/4) and (UCT/CSSR/2019/01), Oxford University (CUREC2/12‐21), provincial Departments of Health and Education, and ethical review boards of participating healthcare facilities.2.1– past‐week adherence – was defined based on indicators of currently taking ART and not having missed any doses in the past seven days (including weekdays and weekends) . Support for the authors and team is provided by the UKRI GCRF Accelerating Achievement for Africa's Adolescents Hub (Grant Ref: ES/S008101/1), the European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Programme (grant agreement no. 771468), UNICEF Eastern and Southern Africa Office (UNICEF‐ESARO), Oak Foundation (R46194/AA001 & OFIL‐20‐057), the Fogarty International Center, National Institute on Mental Health, National Institutes of Health (Award Number K43TW011434). This study was also possible thanks to the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement, and by the Department of Health Social Care (DHSC) through its National Institutes of Health Research (NIHR) [MR/R022372/1], University of Oxford's ESRC Impact Acceleration Account (IAA) [K1311‐KEA‐004 & 1602‐KEA‐189] and Clarendon‐Green Templeton College Scholarship, the Economic and Social Research Council [IAA‐MT13‐ 003], the John Fell Fund [103/757 & 161/033], the Leverhulme Trust [PLP‐2014‐095] and Research England.The content is solely the responsibility of the authors and does not necessarily represent the official views of the agencies, institutions or organizations listed.ET and LC designed and implemented the overall study. LC conceptualized the analyses. SZ led the analyses and write‐up of the manuscript. LC, ET, YS provided edits and feedback on manuscript content and have approved the final draft.The authors declare that they have no competing interests.Table S1: Comparison of baseline indicators for complete and missing viral load measurementsTable S2: Comparison of baseline characteristics for complete cases and LFTU* (Full sample)Figure S1: Wave‐specific past‐week adherence rates among adolescents living with HIV (N=933)Click here for additional data file.


There is a growing recognition of the impact of gender and the social determinants of health on the clinical course of people living with HIV (PLHIV). However, the relative contribution of these factors to clinical outcomes of PLHIV is incompletely defined in many countries. This study was performed to gain a greater understanding of the non-clinical determinants of prognosis of PLHIV in Myanmar.Selected demographic, behavioural and socioeconomic characteristics of outpatients at two specialist HIV hospitals and one general hospital in Yangon, Myanmar were correlated with their subsequent clinical course; a poor outcome was defined as death, hospitalisation, loss to follow-up or a detectable viral load at 6 months of follow-up.3, 138 (62.4%) were male. Socioeconomic disadvantage was common: the median (interquartile range (IQR) monthly per-capita income in the cohort was US$48 (31–77); 153 (69.9%) had not completed high school. However, in a multivariate analysis that considered demographic, behavioural, clinical factors and social determinants of health, male gender was the only predictor of a poor outcome: odds ratio : 2.33 . All eight of the deaths and hospitalisations in the cohort occurred in males (p = 0.03).221 consecutive individuals with advanced HIV commencing anti-retroviral therapy (ART) were enrolled in the study; their median CD4 T-cell count was 92 (44–158) cells/mmMen starting ART in Myanmar have a poorer prognosis than women. Expanded implementation of gender-specific management strategies is likely to be necessary to improve outcomes.The online version contains supplementary material available at 10.1186/s12981-021-00364-w. This isGender also has a significant impact on the clinical outcomes of PLHIV . GloballThis pilot study was performed to determine the demographic and socioeconomic characteristics of PLHIV in Myanmar and the contribution of these factors to short-term morbidity and mortality. The study’s objective was to gain a greater understanding of the non-clinical determinants of prognosis to inform a more comprehensive approach to the local care of PLHIV.This study included participants of a randomized controlled trial (RCT) which was being performed at 3 ART clinics  in Yangon, Myanmar’s largest city.3 or WHO stage 3 or 4 disease), with no contraindication to IPT , peripheral neuropathy or allergy to isoniazid).The RCT examines immediate versus deferred isoniazid preventive therapy (IPT) in PLHIV with advanced disease. The RCT commenced in January 2018 and is continuing at the time of writing. Participants were eligible for the RCT if they were ART naïve adults (≥ 18 years) with advanced HIV (CD4 cell count < 200 cells/mmpro forma. Household income data were collected in Myanmar Kyat (MMK), but are expressed here in United States Dollars (USD) using an exchange rate of 1 USD to 1300MMK. The participants were then followed sequentially. The primary endpoint for the study was a poor clinical outcome, which was defined as death, hospitalisation, loss to follow-up or a detectable viral load (≥ 40 copies/mL) at 6 months.RCT participants were identified in the outpatient ART clinics of the three study sites and enrolled into the study after written, informed consent. Between January and October 2019, participants were also invited to participate in this study. After study doctors obtained written, informed consent for this substudy, demographic, socioeconomic, and selected clinical data were collected using a dedicated Data were de-identified, entered into an electronic database .There were 221 patients enrolled into this study, their median  age was 37 (32–46) and 138 (62.4%) were male. Their baseline characteristics and clinical course, stratified by gender, are presented in Table The demographic and socioeconomic characteristics of the men and women were similar. The median (IQR) age of the men was 37 (31–46) compared with 36 (33–44) among women (p = 0.95). A similar proportion of men and women were unmarried (66/138 (47.8%) versus 40/83 (48.2%), p = 0.95), a similar proportion had completed high school (43/136 (31.6%) versus 23/83 (27.7%), p = 0.54) and a similar proportion lived outside Yangon (38/138 (27.5%) versus 16/83 (19.3%), p = 0.17). The number of people living at home was comparable for men and women (median (IQR): 3 (2–4) versus 3 (2–5), p = 0.90) as was the monthly per-capita income (median (IQR): USD 46.15 (30.77–76.92) versus USD 51.28 (28.04–76.92), p = 0.90).However, men were more likely to be employed than women (119/138 (86.2%) versus 49/83 (59.0%), p < 0.0001). Men were also more likely to report hazardous alcohol use (47/137 (34.1%) versus 0/83, p < 0.0001), more likely to smoke tobacco (54/128 (42.2%) versus 2/83 (2.4%), p < 0.0001) and to chew betel (70/118 (59.3%) versus 7/82 (8.5%), p < 0.0001). The difference in intravenous drug use (3/138 (2.2%) versus 0/83) did not reach statistical significance (p = 0.29).2, p = 0.29) as was the mid-upper arm circumference (MUAC) (25 (22–27) versus 25 (22–27) cm, p = 0.99). The median CD4 cell count was comparable (92 (41–157) versus 92 (48–158) cells/mm3, p = 0.61), however men had less advanced clinical disease at enrolment (median (IQR) WHO stage 1 (1–3) versus 2 (1–3), p = 0.02) and a higher haemoglobin level (median (IQR) 12 (10–13.2) versus 10 (9–11) g/dL, p = 0.0001).The median (IQR) Body Mass Index (BMI) of men and women was similar (19.3 (17.2–22.2) versus 18.9 (16.1–22.3) kg/mOf the 221 patients enrolled in the study, there were 40  patients who were not able to have 6-month viral load testing; in 13 (32.5%) this was due to the evolving global COVID-19 pandemic which prevented clinic attendance for testing. Although none of these 40 patients died, were hospitalised, or were lost to follow-up, the absence of follow-up virological assessment precluded analysis of these patients using the pre-specified primary endpoint.Among the remaining 181 patients, 94 (52%) had a poor outcome  versus 0/83 women, p = 0.03), but there was no gender difference in loss to follow-up (10/138 (7.3%) versus 6/83 7.2%), p = 1.0). A detectable viral load was also more common in men than women (55/90 (53.5%) versus 20/61 (23.8%), p = 0.01). Gender was the only predictor of a poor outcome in univariate analysis  per-capita monthly income was only USD 48 (31–77)) in a country where the median per-capita monthly income is USD 51 [In contrast, the association between gender and outcomes seen in the cohort was striking. The association between male gender and poor outcome in PLHIV is consistent with previous work performed in Myanmar and other countries , 11–13. This is a pattern seen in numerous health conditions, not just HIV. Despite the fact that in many countries men generally have significantly greater privilege and opportunity than women, this advantage does not necessarily translate into better health outcomes . The mosIt is estimated that 87% (78–95) of women ≥ 15 are receiving ART in Myanmar compared with 70% (62–80) of men ≥ 15 years of age . In a coEven though the study only enrolled patients with advanced disease—who were generally profoundly socioeconomically disadvantaged—six months after starting ART, less than 3% had died and less than 7% lost to follow up. This suggests that once patients in Myanmar are able to access ART they usually do well. This may be explained by the fact that all HIV care in Myanmar is free of charge, that there is increasing access to contemporary ART (over 80% of this cohort received a dolutegravir-based regimen) and that medication adherence is generally excellent . The GloThis study has limitations. The lack of association between the social determinants of health and outcomes seen in this small cohort (which increases the likelihood of a type 2 error) does not suggest that these factors can be ignored. There is a significant body of work that demonstrates their importance and larger studies have shown their impact on HIV-related outcomes , 11, 24.Socioeconomic disadvantage was common in this cohort of PLHIV in urban Myanmar, however conventional SDH were not associated with important clinical outcomes during 6-months of follow-up. Gender had more prognostic utility and is likely linked to health-related behaviours that result from community level gender norms in Myanmar. Addressing and modifying these gender norms is challenging, however these data demonstrate that health care workers in Myanmar may need to provide HIV care that recognizes the contribution of gender to the clinical course if they are to deliver optimal outcomes.Additional file 1. Dataset.


Antiretroviral treatment has undergone major changes in the last 20 years, from monotherapy, to dual therapy and finally to triple therapy. Lately, more focus has been placed on better, more well-tolerated combinations and formulations. As in most other disciplines in medicine, the development of paediatric HIV dosages and formulations always tends to lag behind adult research. Twenty years ago, it could take several years before data were available to enable the use of life-saving antiretrovirals in children. Paediatricians, being ever resourceful, were not prepared to let their paediatric patients suffer despite the lack of data or formulations and so made a plan. This article describes some of the trials and tribulations that we went through trying to make sure that our paediatric HIV patients not only survived but thrived. Clinicians treating paediatric patients today have it so much easier because of what our colleagues and their patients went through in those early days. It is hard to convey the sense of helplessness felt by those of us who worked in the pre-antiretroviral treatment (ART) era when faced with a sick HIV-infected child and mother. We had little to offer except the treatment of opportunistic infections and co-trimoxazole prophylaxis. We have come a long way since then.New England Journal of Medicine suggesting that didanosine (ddI) monotherapy was as good as the combination of zidovudine (AZT) and ddI and better than AZT alone.1 At the time, viral load monitoring was unavailable, and the less useful marker of HIV disease progression and/or death was used in clinical trials. Because ddI monotherapy was quite affordable (R80.00 a month for a baby), a few patients in the state sector were able to afford ddI monotherapy, and this did provide a brief respite from the ravages of the disease.In the mid-1990s, when HIV treatment had evolved only as far as dual nucleoside reverse transcriptase inhibitors (NRTIs), an article appeared in the 3In 1998, two developments spurred the cause of paediatric HIV treatment in South Africa. The first was the launch of stavudine (d4T) in South Africa. We now know that this is a very toxic drug, but in those days, it was heralded as a new, very potent NRTI. However, no matter how low the required paediatric dosage, d4T remained expensive and often unaffordable, at approximately R1000.00 a month. To help our patients, one of us would purchase a month’s supply of the adult 40 mg capsules costing R1000.00 and have a chemist split the contents of the capsule into two and put each half back into another capsule. That way, the 20 mg capsules were made available at half price. Stavudine powder for reconstitution with water to form a suspension of 1 mg/mL and dosed at 1 mg/kg per dose twice a day was subsequently released but required refrigeration and the administration of relatively large volumes of liquid medication to young children. To get around these problems, many clinicians treating children unable to swallow capsules or in whom a lower dose was required used a technique of opening d4T capsules, dispersing the powder contents in water and administering an appropriate weight-based dose. Some years later, pharmacological and pharmacokinetic evaluations using high-performance liquid chromatography confirmed the accuracy of the off-label opened-capsule dosing method for stavudine and showed that plasma drug exposure after stavudine administration as a solution in this way was bioequivalent to intact capsule administration.5 However, its toxicity – lactic acidosis, often with fatal consequences – was quickly recognised, and this therapy ended.However, ARVs were still very expensive, and few individuals could afford them. They were not available in the state sector, and medical aids were generally not paying for them, either. In 1998, Aid for AIDS (AfA) was launched to assist medical insurance schemes to manage HIV in the private sector. It was revolutionary, but there was a limit – adult patients could only receive dual therapy and not triple therapy. One of the researchers (L.J.L.) approached AfA (Dr Leon Regensburg) and asked if it would be possible for paediatric patients to receive triple therapy, because they could get three drugs for the same price as two drugs in adults. AfA did, as a result, allow paediatric patients triple therapy; this is why some paediatric patients in the private sector received triple therapy as early as 1998. Although many were on d4T/ddI/ritonavir (RTV) – not a great regimen in anybody’s book – many survived and are alive and well today. While paediatric ARV experience was evolving in the private sector, nothing was available in the state sector. Hydroxyurea, a cheap drug used in oncology and believed to be synergistic with ddI, was tried. Because of its low cost, patients, including children, were treated with the combination.Southern African Journal of HIV Medicine. In the early 2000s, very limited access to paediatric ART in the public sector was mostly via international humanitarian non-governmental organisations such as Médecins sans frontiers (Doctors Without Borders) and donor-supported hospital-based programmes.7 On 01 April 2004, the national ARV roll-out began.8 This was wonderful, but paediatric formulations were far from optimal. Children were given d4T/lamivudine (3TC) combined with lopinavir/r (LPV/r) if they were under 3 years of age or efavirenz if they were over 3 years. Besides the problem of the terrible taste and poor tolerability of LPV/r, it was not used in children under 6 months in the absence of data on its use in this age group. So, any child under 6 months was given full-dose RTV, as were any patients on concurrent rifampicin-containing antituberculosis treatment. Besides the bad taste of RTV, this protease inhibitor (PI) presents HIV with a low genetic barrier to resistance, resulting in many children developing PI resistance.9 Several years later, the NRTI backbone was changed to abacavir and 3TC, a safer combination and one that allowed for once-daily dosing – a big help in terms of adherence.From 2000, things changed for the better. The AIDS 2000 conference was a watershed moment, as was the launch of the One of the obstacles to achieving widespread coverage of paediatric ART has been the simplification of the prescribing process for non-paediatricians, including doctors and nurses who are more familiar with prescribing ART for adults but who are also involved in treating children, particularly at the primary care level. Calculating individualised doses of separate and mostly liquid oral ARV formulations for an infant or young child at each clinic visit using the current weight or body surface area is complicated and time-consuming. The development and updating of an integrated weight-based ARV dosing chart for children based on World Health Organization guidelines and adapted for the ARV formulations available in South Africa has contributed to building confidence amongst prescribing clinicians and pharmacists and helped facilitate children’s access to ART.10 In the 2019 national guidelines, tenofovir/3TC/dolutegravir fixed-dose-combination tablets can be used from 10 years of age and 35 kg upwards,11 making a well-tolerated single-tablet regimen available for the first time to these adolescents. Another issue that is still a problem is adolescent disclosure, because very few healthcare workers feel comfortable disclosing to their adolescent patients, and parents are obviously petrified to do so because of feelings of guilt and worries that the children may be angry with them. This and other adolescent issues are resulting in very low rates of adolescent viral suppression.12However, issues of ARV tolerability and access to formulations appropriate for children remain. The LPV/r formulation is very unpleasant to taste, and whilst young babies tend to tolerate it when their taste buds are still undeveloped, as they grow older, they often spit or vomit it out. Only in 2020 did the LPV/r pellets become available both in the state and private sectors. However, in the state sector, it may only be prescribed for patients over 6 months of age who are not tolerating the LPV/r solution.In South Africa, HIV viral resistance testing first became available in the early 2000s. At the time, a prevailing practice was to keep children with high viral loads on the same ART regimen as long as the CD4 count was acceptable. This was a pragmatic approach because of the limited treatment options available and the restricted access to resistance testing but is likely to have contributed to the accumulation of progressive resistance mutations in some children, thereby limiting future treatment options. A national third-line ART expert committee was established in 2013, focusing on patients with virological failure on a PI-based ART regimen. New-generation PIs and the integrase strand transfer inhibitor (INSTI) class of ARV drugs became accessible, though on a limited basis. This has provided real hope for treatment-experienced adults and children and their clinicians in achieving better long-term treatment outcomes.13 culminating in January 2015 in the provision of lifelong triple ART for all pregnant and breastfeeding women. Whilst fewer children are acquiring HIV through vertical transmission, we are finding these children particularly challenging to deal with. Pregnant women who know their HIV status and are able to take ART rarely transmit HIV to their babies. Of course, some mothers acquire HIV for the first time during their pregnancy or whilst breastfeeding and have unfortunately already transmitted the infection to their baby before they are aware of their status. However, the majority of infected babies in the era of ART are born to mothers who for complex social reasons have defaulted on their ART or take it inconsistently. Poverty, stigma, and drug and alcohol abuse are all contributory factors. Unfortunately, many of these mothers in turn find it difficult to give ARVs to their children and to access treatment for them. Many of the children who acquire HIV now are born into families with the least ability to deal with it. The hopelessness we felt in the pre-ART era is now replaced by the heartbreak and frustration of having life-saving treatment that is not taken.Alongside the changes in paediatric ART, the reduction in mother-to-child transmission (MTCT) resulting from maternal ART has been dramatic. Between 2009 and 2015, new paediatric infections decreased by 84% as a result of our national prevention of MTCT guidelines evolving rapidly,In summary, paediatric ART in South Africa has come a long way: monotherapy, dual therapy, toxic regimens with d4T, ddI, and RTV. However, a universally available, well-tolerated, single formulation available from birth to adolescence still eludes us. Whilst advocating for new, simpler and better-tolerated paediatric regimens, an underlying constraint remains – that of improving the support of vulnerable patients, especially pregnant and breastfeeding mothers, so that they are able to take ART consistently and ensure the protection of the next generation of South Africans.


There is poor viral load monitoring (VLM) and inadequate management of virological failure in HIV-positive individuals on antiretroviral therapy in rural KwaZulu-Natal, South Africa. This could be contributing to increasing HIV drug resistance in the setting. This study aims to investigate the clinical and process impediments in VLM within the health system and to evaluate a quality improvement package (QIP) to address the identified gaps. The QIP comprises (i) a designated viral load champion responsible for administrative management and triaging of viral load results (ii) technological enhancement of the routine clinic-based Three Interlinked Electronic Register (TIER.Net) to facilitate daily automatic import of viral load results from the National Health Service Laboratory to TIER.Net (iii) development of a dashboard system to support VLM.The study will evaluate the effectiveness of the QIP compared to current care for improving VLM and virological suppression using an effectiveness implementation hybrid type 3 design. This will use a cluster-randomised design with the primary healthcare clinics as the unit of randomisation with ten clinics randomised in a 1:1 ratio to either the intervention or control arm. We will enrol 150 HIV-positive individuals who had been on ART for ≥ 12 months from each of the ten clinics (750 in 5 intervention clinics vs. 750 in 5 control clinics) and follow them up for a period of 12 months. The primary outcome is the proportion of all patients who have a viral load (VL) measurement and are virally suppressed (composite outcome) after 12 months of follow up. Secondary outcomes during follow up include proportion of all patients with at least one documented VL in TIER.Net, proportion with VL ≥ 50 copies/mL, proportion with VL ≥ 1000 copies/mL  and subsequent switch to second-line ART.We aim to provide evidence that a staff-centred quality improvement package, designated viral load monitoring champion, and augmentation of TIER.Net with a dashboard system will improve viral load monitoring and lead to improved virological suppression.Trial registration: This trial is registered on ClinicalTrials.gov on 8 Oct 2021. Identifier: NCT05071573; https://clinicaltrials.gov/ct2/show/NCT05071573?term=NCT05071573&draw=2&rank=1 Antiretroviral therapy (ART) scale-up in sub-Saharan Africa (SSA) has been rapid with an estimated 16.4 million individuals on ART by the end of 2018 . In settSouth Africa has the biggest HIV treatment programme in the world with 7.7 million individuals living with HIV and 62% currently receiving ART within a stretched health system . VLM hasHowever, little is known about how these VL guidelines are being used in clinical decision-making in public ART programmes in SSA. In formative research utilising an electronic database, the Three Interlinked Electronic Register (TIER.Net) , of a prFindings from this formative research was presented in a series of workshops to healthcare providers, policy makers, researchers and community representatives using good participatory approaches with a view to co-produce health system interventions to address poor VLM. This protocol describes the proposed interventions and their evaluation. We hypothesise that a staff-centred quality improvement package (QIP) and technological augmentation of an existing electronic ART database (TIER.Net) would result in optimal VLM of patients on ART, prompt clinical management of virological failure and an overall improvement in virological suppression.The main objective is to evaluate the impact of a combination of interventions that includes a staff-centred quality improvement package, designated viral load monitoring champion, and augmentation of TIER.Net with a dashboard system will improve viral load monitoring and lead to better virological suppression over a period of 12 months in comparison to the current standard of care.To identify health system specific gaps in VLM.To evaluate the cost and cost effectiveness of the intervention compared to standard careTo undertake a process evaluation assessing acceptability, fidelity, adaptation and contexts in the implementation of the intervention.The trial will be hosted by the Africa Health Research Institute (AHRI), an independent scientific research institute and a Wellcome Trust Africa and Asia Programme. AHRI has two campuses 250 km apart; the Population Intervention Programme (PIP) study area in uMkhanyakude district in northern KZN with a population of 625,846, and a state-of-the-art laboratory infrastructure in the city of Durban.2. Hlabisa is at the epicentre of the HIV epidemic in South Africa, where HIV prevalence among men and women aged 15–54 years in 2018 was 19% and 40% respectively [The PIP is an extensive social, demographic, HIV and clinical research programme of an under-resourced rural population of 140,000 in an area of about 845 kmectively . It is sectively . AHRI haThe study involves collaboration of investigators from AHRI, the University of Sussex (UoS) and the University of Cape Town (UCT).We will use a retrospective chart review of HIV positive patients in care at the 10 trial clinics to identify the health systems gaps in VLM. We will create sampling frame of individuals who are in care and started ART 15–18 months previously and select a random sample of 800 individuals from this frame. Clinical notes of these individuals will be reviewed to identify any gaps in the VLM process, and to identify predictors of missing viral loads. This information will be used to develop a QIP that includes training of staff on the current South African ART guidelines, the importance of VLM and both health system and individual risk factors for suboptimal VLM.We will use an innovative effectiveness-implementation hybrid cluster-randomised trial in the 10 clinics to evaluate the effectiveness of the interventions for improving VLM and virological suppression compared with current care .The proportion lost to follow-up.Those with confirmed virological failure (repeat VL ≥ 1000 copies/mL after a first VL 1000 copies/mL measured ≥ 3 months apart).As part of the proposed intervention, VL results will be imported into TIER.Net daily from the National Health Laboratory Service (NHLS) which will be linked to patients in TIER.Net based on multiple exclusive and linked deterministic rules using a combination of variables such as name, surname, sex, date of birth, date of visit, NHLS lab number, facility and folder number. The information contained in TIER.Net will be used to develop a dashboard which will display the following information:A dashboard has the advantage of displaying a quick visual state of affairs and how this evolves with time. This will incorporate a text messaging functionality that can be used by healthcare workers/viral load champion to communicate with patients.It is envisaged that these interventions will result in prompt identification of patients who require VLM, and improve clinical management of virological failure thereby improving the quality and efficiency of service delivery.Viral load results are manually captured on to the TIER.Net system with filing of the paper results in patients’ clinical notes for nurses’ review during routine appointments. This is used to produce a monthly enrolment and quarterly cohort reports for the central monitoring of the ART programme.Proportion of patients in survey sample with VL documented in clinical charts but not in TIER.NetProportion of patients with VL documented in TIER.Net but no results in clinical chartsProportion of patients with no VL in chart or TIER.Net (true missing VL = no assessment of VL undertaken)Predictors of true missing viral load Proportion of all patients who have a VL measurement and are virally suppressed (composite outcome) after 12 months of follow up.Proportion of all patients with at least one documented VL in TIER.Net during the trial follow up.Proportion with VL ≥ 50 copies/mL during follow upProportion with VL ≥ 1000 copies/mL during follow up Proportion with a repeat test within 3 monthsTime from first VL ≥ 1000 copies/mL to repeat VL.Proportion switching to second-line ART after two consecutive VL ≥ 1000 copies/mL measured ≤ 3 months apartProportion completing the treatment failure cascade (VL < 50 copies/mL on repeat testing or switch to second line ART)Among patients with VL ≥ 1000:Cost-effectiveness of the intervention compared to current care with respect to the primary outcome  and two secondary outcomes (proportions of patients with VL ≥ 50 copies/mL and VL ≥ 1000 copies/mL)Acceptability, fidelity, adaptation and contexts in the implementation of the interventionsThe precision with which the prevalence of each outcome can be estimated will depends on the prevalence itself and the amount of correlation within clinics (the ‘design effect’). For a prevalence of 30%, a sample size of 800 patients will allow us to estimate it with a precision of ± 4.5 or ± 5.5% with 95% confidence if the design effect is 2 or 3, respectively. For a prevalence of 60%, the corresponding figures are ± 4.8 and ± 5.9%. This sample size will also allow us to determine the association of factors with a missing VL measurement. For example, if the design effect is 2, assuming the prevalence of missing VL is between 30 and 60%, we would have > 80% power to detect an OR of 2.25 or more for factors associated with missing VL, if the prevalence of the risk factor among those with non-missing VL is between 15 and 70%. This would allow us to identify any strong predictors of missing VL.We estimate that during the 3 month recruitment phase, an average of 150 patients who have been on ART for ≥ 12 months will attend each clinic. Based on our previous work, we expect that around 50% of patients in the control arm will have a VL measurement and be virally suppressed at the end of follow-up. With 5 clinics per arm, assuming a coefficient of variation, k, of 0.20, and a sample size of 150 patients per clinic, we would have 80% power to detect an increase in the proportion of patients who have a VL measurement and are virally suppressed from 50% in the control arm to 77% in the intervention arm .AHRI has a clinical research assistant stationed in each of the ten clinics who registers every patient attending the clinic, irrespective of their reason for attendance or HIV status. A database, known as ClinicLink, was developed in-house at AHRI for the registration of clinic visits and reasons for attendance. ClinicLink will be used to identify patients attending the clinic who meet the criteria for inclusion in the evaluation cohort. Based on current clinic attendance, we will need about three months to meet the target sample size of an average of 150 patients per clinic. These individuals will form the basis for the main analyses. In secondary analyses, all patients meeting these criteria who attend one of the trial clinics during the three-month recruitment phase will be included in the evaluation cohort, even if the sample size is exceeded since the target sample size is considered a minimum. If the target sample size is not met within 3 months, we will extend the recruitment phase for another month as necessary.The 10 clinics will be randomly allocated to the intervention (5 clinics) and control (5 clinics) arms. Randomisation will be stratified on clinic size, defined by the number of HIV-related visits per month (2 strata). Restricted randomisation will be used to ensure balance between study arms on the following important covariates: proportion of HIV patients who are male; proportion of HIV patients who are aged < 25 years. A computer programme will be used to prepare a list of all permissible randomised combinations; community leaders will be invited to make a random selection from the list at a public ceremony. The randomisation list will be prepared by an independent AHRI statistician and will be concealed until after randomisation. It will not be possible to blind the research staff and nurses to the intervention.A secure Research Electronic Data Capture (REDCap)  project Before data collection begins, the REDCap will be thoroughly tested and subjected to a range of checks culminating in the entry of data for a group of ‘dummy’ patients, and this will be followed by extraction of dummy dataset by a research data manager and derivation of key study outcome measures by the study statistician to ensure data fields and response coding are clear. After all checks, the database is ‘signed off’ by the research data manager and statistician.Once data entry has begun periodic validation and management checks will be performed using Pentaho Data Integration to assess data completeness, rate of recruitment and data consistency. Exception reports will be created, and issues raised will be queried with the study coordinator.The following data will be extracted from chart review into REDCap to document if (i) viral load was actually not measured as evidenced by a lack of documentation of this in the clinical charts and in TIER.Net (ii) there was documentation of a blood draw for VL and the date of the test (iii) results were filed and entered into the patient charts although not captured in TIER.Net (iv) there was evidence in patient chart that test was done but no results filed (v) VL results and date present in TIER.Net but not filed in patient charts.All data collected from clinical chart reviews will be centrally stored in a master MONART database located at Africa Health Research Institute. This database will integrate data collected from clinical charts reviews, hospital information management system (HIS), ClinicLink and TIER.net. Database rules and constraints will be added and enforced on the master database to ensure that only valid data is stored in the database.A study dashboard will be developed to assist the operational teams monitor the progress of the study.TIER.Net has information on HIV-positive individuals on ART including unique South African Identification number, age, sex, date of ART initiation, viral load results and dates, CD4 count results and dates, type of ART regimen, status of the individual , and dates of clinic appointments. The clinical charts of the 150 participants per clinic who are in the evaluation cohort will be reviewed to extract these baseline data for entry into REDCap. These will be repeated after each participant has completed 12 months of follow up in their clinic/cluster to capture documentation of VL being measured, test data and the results of the VL. These data will be used to evaluate the impact of the intervention on VLM and virological suppression, and on the treatment failure cascade. Data on all clinic visits irrespective of reason for the visit will be extracted from ClinicLink. These will be used to describe patterns of health care use among the trial population. HIV-positive individuals receiving care in the clinics are not the recipients of the quality improvement interventions, which will be focussed on the nurses and systems for delivering care. Hence, interim visits for any intercurrent illness by patients will be managed as per standard clinical care and will not be routinely reported as adverse events.We will pilot the interventions over 3 months to address any issues raised and deliver further training to the nurses or adaptation of the technology if required Fig. . PatientDue to data protection regulation, AHRI will act as data controller for the project. UCT and UoS will be data processors but will only process anonymised data for the purpose of analysis. It will be the responsibility of the data custodian based at AHRI to ensure that data access is consistent with terms of the AHRI Data Access Policy and ethical approval obtained for this protocol.Data will be stored on industry-standard relational databases with data integrity and user authentication for access control. Data will be replicated on at least a daily basis to the Durban site of the Institution to provide secure offsite storage of data. Transactional logs will be backed up every 30 min to enable recovery of data in the event of equipment failure.All users of the MONART electronic data collection system (REDCap) and database will be authenticated through individual passwords with minimum complexity and regular change rules . The Institution use industry standard malware and intrusion detection with regular penetration tests by a reputable external security audit company.Both at the Institute and for the clinic-based data collection, a client–server architecture will be implemented where data is not stored on laptops or local workstation, but only on a central server with restricted physical access. Specifically, at the clinics the local server will be enclosed in a tamper-proof enclosure kept under lock and key.Once data have been stored in the MONART databases, they will become the responsibility of the head of research data management. Research data management will regularly assess data quality and implement corrective measures to ensure data quality. At the end of the study, anonymised analytical datasets will be created, documented and archived on the AHRI data repository. A digital object identifier (DOI) will be created and added on the dataset documentation for reference purpose. Research data management will administer a data repository that will contain all datasets required for analysis for the trial. They will ensure that the repository has access control measures that enforce the data access criteria determined by the study data steering committee. Access to the datasets will be granted to those who meet minimum requirements set out by the study data steering committee.All correspondence relating to this project will be kept in appropriate file folders. Any confidential patient or site information will be kept in secure, limited access locations. Records of participants, other source documents, pertaining to the study will be kept on file for a period of 10 years per South African Good Clinical Practice guidelines (SA GCP).Analysis of data from the retrospective chart review of patients will quantify clinical and process impediments in VL testing system; results will inform the VL testing pathway, a QIP and required augmentation of TIER.Net. Proportions of patients with outcomes of interest will be tabulated; 95% confidence intervals will be calculated using clustered standard errors to take correlation within clinic into account. Factors associated with true missing viral load (not documented in TIER.Net or present in the patient chart) will be analysed using logistic regression with robust standard errors.The effect of the intervention on the primary outcome (proportion of patients with VL suppression) will be estimated using a two-stage approach based on cluster (i.e. clinic)-level summaries . The cluIt was not relevant to set up a data monitoring and safety board as routine health data will be assessed for study outcomes following the quality improvement intervention delivered to research nurses and augmentation of TIER.Net. Hence, we decided to set up a trial steering committee in addition to the trial management group.The trial management group will include the principal investigator and all the co-investigators including study statistician, research data manager, study coordinator and the operational managers of the five intervention clinics. The TMG will be chaired by the principal investigator and will have day to day oversight responsibility for the conduct of the trial.They will meet once a month either in person or virtually to discuss the progress of the trial. They will also review all data from the trial and will be the main decision making body of the trial. They will be advised by an independent trial steering committee.This will consist of individuals who are independent from the study team. They will be joined by members of the TMG including the principal investigator, an investigator nominated by each collaborating institution, study statistician, study coordinator and research data manager. The composition will be specified in the TSC charter. The TSC will have operational responsibility for the overall conduct and management of the trial. The group’s terms of reference, roles and responsibilities will be defined in a standard operating procedure. One of the independent members of the TSC will act as the chair.This study is registered with Clinicaltrials.gov (NCT05071573). The main study findings will be reported in accordance the Consolidated Standards of Reporting Trials (CONSORT) statement . The stuThe operational managers of the 10 trial clinics will be given clear explanations about the trial aims, and the nature, scope, and possible consequences of their facilities and staff participating in the research by the study coordinator. Managers will be asked to provide written informed consent for their facilities to participate in the trial and be randomised to one of the two trial arms, and for review of clinical records of patients.The principal investigator and study coordinator will schedule meetings with nursing staff from the 10 clinics, to explain the intervention and the trial aims. Nurses will be asked to consent to participation in the trial, and to receive further training as viral load champions if selected. They will be offered consent for participation in the process evaluation (not described in this protocol).The informed consent process will be administered by a research assistant through REDCap with electronic signature obtained from nurses participating in the QIP. The nurses will be given paper copies of the participant information leaflets and consent forms.A waiver of individual written consent from patients for a review of their clinical records is approved by all the regulatory authorities mentioned above. We requested this waiver for the following reasons: (1) the research involves minimal risks to the patients, (2) the extracted data will be anonymised hence there is no risk of the confidentiality of patients being compromised, and (3) analytical results will be reported at population or group level and not at the level of the individual patient.We have a memorandum of agreement with the Department of Health to provide access to records in TIER.Net. Extraction of data from TIER.Net and ClinicLink, and linkage to other AHRI databases, has been previously approved by the University of KwaZulu-Natal Biomedical Research Ethics Committee (Ref: BE290/16).All investigators and trial site staff will comply with the requirements of the Protection of Personal Information Act (or POPI Act)  which ishttps://data.ahri.org/index.php/home. Authorship of research manuscripts will follow the International Committee of Medical Journal Editors guidelines [Research findings will be fed directly back to the community advisory board and the district and provincial departments of health prior to sharing this with the wider scientific community. In addition, under the existing memorandum of agreement between AHRI and the KwaZulu-Natal Department of Health, all scientific work  will be submitted for review by the department of health through the Head of the Health Research Committee. Study staff will feed results back to sites involved in collaboration with the department of health as they emerge and discuss areas that require strengthening. Investigators will publish findings in peer-reviewed journals and present at relevant conferences and meetings. Access to the final dataset will be according to AHRI data sharing policy available at idelines .In the formative research leading up to the interventions proposed for evaluation in this protocol, we showed poor viral load monitoring and inadequate management of virological failure in HIV-positive patients on ART in rural KwaZulu-Natal, South Africa. Non-Nucleoside reverse transcriptase pre-treatment drug resistance exceeds 10% in the setting ; the thrThe observed poor VLM in KwaZulu-Natal could be due to a number of reasons; patients may not have been aware of or empowered to request recommended blood tests, facility staff may be inadequately trained, or have insufficient time or resources to conduct blood tests when due, failure of results to reach clinics from the laboratory or failure to capture results on TIER.Net as these are manually entered . The curHence there is urgent need to evaluate interventions that will improve VLM and the management of virological failure in ART programmes in sub-Saharan Africa. These interventions will have to be cost-effective, acceptable by patients and feasible to adopt in the health system. These additional important objectives will be addressed in this research but are not covered in this protocol. If the combination of interventions proposed for evaluation in this trial demonstrates effectiveness, the resulting early detection and prompt management of virological failure will be critical towards achieving UNAIDS 95-95-95  targets by 2030 .


At T1, 4/6 APACHE and 2/5 MODAt carried HIV-DNA MRMs. MRMs disappeared at T3 in 3/4 APACHE. All disappearing MRMs were characterized by T1 intra-patient prevalence <80%, and mainly occurred in APOBEC3-related sites. All MRMs persisted over-time in the 2 MODAt. C2-V3-C3 genetic-distance significantly changed from T1 to T3 in APACHE individuals , while no significant changes were found in MODAt. Accordingly, maximum likelihood trees (bootstrap > 70%) and genealogical sorting indices  showed that T1 C2-V3-C3 DNA sequences were distinct from T2 and T3 viruses in 4/6 APACHE. Virus populations at all three time-points were highly interspersed in MODAt. Conclusions: This pilot study indicates that short ATI does not alter peripheral-total HIV-DNA burden and residual viremia, but in some cases could cause a genetic diversification of peripheral viral reservoir in term of both MRMs rearrangement and viral evolution.Background: If analytical antiretroviral-treatment (ART) interruption (ATI) might significantly impact quantitative or qualitative peripheral-total HIV-DNA is still debated. Methods: Six chronically HIV-1 infected patients enrolled in APACHE-study were analysed for peripheral-total HIV-DNA and residual viremia, major-resistance-mutations (MRMs) and C2-V3-C3 evolution at pre-ATI (T1), during ATI (T2) and at achievement of virological success after ART-resumption . These data were obtained at three comparable time-points in five chronically HIV-1 infected patients on suppressive ART for ≥1 year, enrolled in MODAt-study. Results: At T1, APACHE and MODAt individuals had similar peripheral-total HIV-DNA and residual viremia ( Antiretroviral therapy (ART) can suppress HIV-1 viremia below the detection limit in clinical assays tests and therefore is able to drastically reduce morbidity and mortality derived from HIV-1 infection . Despite+T-cells with therapeutic agents that target the virus and/or infected cells, and harnessing anti-HIV immune-responses using therapeutic vaccines or immunotherapies [To date multiple strategies aimed at preventing and/or contain the establishment and size of persistent HIV reservoirs have been applied ,9,10. Exherapies ,12. Earlherapies , can limherapies ,15,16, aherapies .Recently, analytical treatment interruptions (ATI) or treatment simplification strategies have become a focus of investigations as innovative approaches to the long-term management of HIV disease. ATIs have been used in different studies towards an HIV cure, as in acute, primary and chronic infection ,19 and iCurrent challenges with ATI include identifying an appropriate timeframe for length of interruptions and identifying HIV patient populations able to spontaneously control viral replication once antiretroviral drug therapy is withdrawn.Nonetheless, concerns remain about the effect of ATI on the HIV-1 reservoir, and on the existence of reliable viral markers able to predict reservoir alterations during/post-ATI ,23,24,25Recent data suggest that even in patients characterized by long period of HIV viral-load suppression and low viral reservoir (APACHE cohort), early and consistent viral-rebound can be observed during ATI . Using s+T-cells >500 cells/mm3, HIV-DNA <100 copies/106 PBMCs by Real-Time PCR  and without comorbidities or AIDS-defining diseases. No patient initiated antiretroviral treatment during acute HIV-1 infection. We analyzed samples for peripheral-total HIV-DNA, residual viremia and HIV variability at three time-points: before ATI , at viral-rebound (VR) during ATI  and at achievement of undetectable viral-load after ART resumption , who underwent ATI. These patients had HIV-RNA <50 copies/mL for ≥10 years, no viral blip for ≥5 years, CD4The same data were obtained at three comparable time-points  in five chronically HIV-1 infected patients treated with a triple-therapy (triple-ART) and on stable virological suppression for >1 year, enrolled in the MODAT study   Figure .Antiretroviral treatment of APACHE and MODAt individuals did not include drugs belonging to integrase inhibitor class (INI).Peripheral blood mononuclear cells (PBMCs) aliquots and plasma samples were collected at all time-points.+T and CD8+T cell count were retrieved and stored in an anonymous database.For all patients, demographic and clinical information such as age, gender, CD4Ethical approval for the APACHE and the MODAt studies were obtained from the Ethical Committee of the San Raffaele . Signed informed consent was obtained by all participants. The APACHE and the MODAt studies were conducted in accordance with the Declaration of Helsinki Ethical Principles and Good Clinical Practices.6 PBMCs by means of the QX200™ Droplet Digital™PCR System  using home-made protocol targeting the LTR-5′ region [6 PBMCs) was then normalized into number copies/106 CD4+T-cells. Residual viremia was quantified from 2 mL of plasma through ddPCR using a home-made protocol targeting Integrase region [Total HIV-DNA was quantified from a pellet of 3 × 106 PBMCs using DNeasy Blood & Tissue kit . Plasma HIV-RNA was extracted using QIamp Viral RNA mini kit (Qiagen). Deep sequencing of POL (HXB2-pol nucleotides:170–1415) and C2-V3-C3 (HXB2-env nucleotides:805–1224) was performed from PBMCs and plasma on MiSeq platform . Briefly, HIV-DNA and HIV-RNA were amplified by PCR with region-specific primers  and quantification by Qubit dsDNA HS-Assay (ThermoFisher), amplicons were pooled in an equimolar concentration and indexed by Kapa-HiFi HotStart . Lastly, 15 pM of denatured pool was sequenced using MiSeq-reagent kit v3  paired end run.All sequences were screened for evidence of baseline quality control for short and low-quality sequences. A quality control was performed by USEARCH commands fastq_filter (using an accepted phred-score of 30)  and unoiIdentical POL sequences  were clustered, allowing identification of most representative strains for each patient at each time point. The output consisting of a list of representative clusters of sequences and their relative frequencies was used to evaluate the presence of major drug resistance mutations (MRMs), hypermutant and thus defective strains, and stop codons.https://hivdb.stanford.edu/, last update 23 October 2020, mutations reported in bold). INI resistance mutations were not considered for this analysis as patients were INI-naïve.MRMs to NRTI, NNRTIs or PI classes were defined according to the Stanford HIV Drug Resistance Database 2020  using clustered and representative POL variants [p-values were estimated for each sequence at each time-point by using as reference both HXB2 and the most abundant strain for each time-point. A p-value < 0.05, confirmed by using as reference both HXB2 and most abundant strain, was used to determine if a specific strain is a hypermutant, and thus probably defective.In order to define a trend of hypermutation and to detect potentially POL defective variants, G-to-A mutation frequencies at GA or GG POL sites potentially induced by APOBEC3-enzymes  and numbvariants . p-valueTo assess potential compartmentalization among sampled virus populations, genetic distance and phylogenetic estimates were performed on C2-V3-C3 region.Firstly, for each sample at each time-point, pairwise genetic distance was computed to quantify nucleotide diversity under the Tamura-Nei 93 model in the total number of obtained C2-V3-C3 strains . Thus, ehttp://molecularevolution.org/software/phylogenetics/gsi) (accessed on 21 November 2020) [To assess potential compartmentalization among sampled virus populations, identical C2-V3-C3 sequences  were clustered, allowing identification of most representative strains for each patient at each time point, following the procedure used for POL sequences. A maximum likelihood tree by using the combinations of clustered C2-V3-C3 proviral-DNA and viral-rebound sequences were inferred using PhyML version 3.0. To assess the relatedness of virus compartments more objectively, we determined genealogical sorting indices (GSI), that tests the phylogenetic similarities of 2 or more sequence groups, with the null hypothesis that the virus populations were the same . GSI valer 2020) . Statistp-values were two-sided and corrected for multiple tests (Benjamini-Hockberg correction) [p-value of less than 0.05 was considered statistically significant. All analyses were performed using statistical software Rgui and SPSS version 19 .All rection) . A p-valp-value = 0.662) for more than 2 years . APACHE individuals experienced viral-rebound between 14 and 56 ATI days and restarted ART after confirmation of two viremia determinations >50 copies/mL. APACHE plasma viral-load was again <50 copies/mL after 95 days (IQR:19–168) of ART restart (median (IQR) residual viremia: 4 (1–9) copies/mL).The demographic and clinical characteristics by APACHE and MODAt participants are shown in MODAt participants remained under virological control for all the study period.To estimate within-person changes in the size of the peripheral latent reservoir pre- and post-ATI, we quantified peripheral-total HIV-DNA from PBMCs by ddPCR for all participants at different time-points.6 CD4+T-cells at T1 (pre-ATI), and 1916 (398–3798) and 1087 (663–1567) copies/106 CD4+T-cells at T2, in APACHE and MODAt subjects, respectively. At T3, the median (IQR) HIV-DNA was 1333 (553–2183) in APACHE-group and 1104 (527–1598) in the MODAt-group.Firstly, no significant differences were observed in peripheral-total HIV-DNA values at T1, T2, and T3 between APACHE and MODAt subjects. In particular, median (IQR) peripheral-total DNA levels was 1048 (863–1286) and 1037 (730–1750) copies/10p-value = 0.004). No difference was detected at T3-T2 .Regarding change in time, time between T2-T1 was significantly lower in APACHE respect to MODAt participants , confirming the decay of HIV-1 reservoir against time [p-value = 0.354).To further evaluate if the modification of HIV-DNA levels observed in APACHE individuals might be affected by the duration in weeks between time-points, ΔHIV-DNA  were compared against Δweeks . Of note, longer was the change in time at T3–T2, higher was the change in HIV-DNA observed at T3–T2  for APACHE, and 58.2% (IQR: 47.1–99.5)  copies/106 CD4+T-cells) for MODAt individuals  .At T2, HIV-DNA MRMs persisted in 2/4 (50.0%) APACHE individuals, with an intra-patient prevalence that decreased from pre-ATI to ATI .At T3, HIV-DNA MRMs persisted only in 1 out 4 APACHE individuals  were found only in 1/6 APACHE sample (ID: A009) . Of note, the two APOBEC3-related mutations at drug-resistance positions characterizing this patient (D67N: 6.2% and E138K: 7.3%) were present in the hypermutant POL strains, thus suggesting the presence of this mutation in potentially defective proviruses. Accordingly, these variants were never detected at following time-points (T2 and T3). Interestingly, at T1 other 3 APACHE individuals carried APOBEC3-related mutations at drug-resistance positions  .p-value = 0.04), while no significant difference was found in MODAt patients . These results were also confirmed by comparing with Friedman tests the C2-V3-C3 genetic distance at the three time-points . Moreover, by applying nonparametric tests to the longitudinally collected samples for each patient, a significant variation in genetic diversity from T1 to T3 was found for 4/6 APACHE individuals, with the sole exception of A001 and A009, and 1/5 MODAT individuals  APACHE patients pre-ATI DNA sequences were generally distinct (boostrap > 70%) from viral-rebound and post-ATI viruses , and thaOn the contrary, in 4/5 (80%) MODAt patients the phylogenies for C2-V3-C3 regions showed that virus populations at the three time-points were highly interspersed .Furthermore, to assess the relatedness of virus compartments more objectively, we determined a genealogical sorting index (GSI). GSI values range from 0 (complete interspersion) to 1 (complete monophyly), with statistical significance indicating greater than random segregation between/among groups.p-value < 0.05). Only the sequences for participants A001 and A009 had low GSI value, indicating similar sequences between T1 and T3 for A001 and highly interspersed sequences over-time for A009  from T2 , suggesting that the rebound viruses during ATI does not arise from the pre-ATI sequences. In four out of six participants, both the T2 proviral and rebound sequences did not differ significantly  from T3 proviral-DNA, suggesting a contribution of rebound sequences to viral evolution of post-ATI viruses.In APACHE patients, GSI values obtained at different time-points indicated that the majority of participants had highly monophyletic virus populations . Indeed, low and no significant GSI values between T1 and T3 were found in 80% (4/5) MODAT patients. Only patient M66 had a statistically significantly high GSI value, indicating changes in the composition of the proviral-DNA over-time  [Here, we used samples from patients enrolled in the APACHE study  to assesression) .p-value = 0.311, +T-cells, as well as immune markers, during ATI [We firstly assessed the impact of ATI on the amount of peripheral-total HIV-DNA, considered as a global marker of HIV persistence and progression ,40. We dring ATI .Even if total HIV-DNA quantification reveals a stability in the amount of peripheral HIV reservoir, differences in the archived MRMs between pre- and post-ATI were found. In particular, in three out four APACHE individuals , pre-ATI MRMs were lost during virological rebound and in post-ATI HIV-DNA. All these MRMs were characterized by pre-ATI intra-patient prevalence <80%. The only APACHE individual where a consistent disappearance of MRMs was not observed (A001), was mostly characterized by MRMs with an intra-patient prevalence >80%. Overall data suggest that the absence of ART selective pressure during ATI combined with the low intra-patient prevalence of some MRMs might participate in the disappearance of low-abundant drug resistance mutations. In line with the hypothesis that ATI and the absence of ART selective pressure might cause a re-assortment of MRMs, in MODAt patients drug resistance mutations persisted across all three time-points independently by their baseline intra-patient prevalence.Interestingly, most of mutations at drug resistance positions found in APACHE subjects  derived by G to A substitutions, and thus probably related to an hypermutation process induced by APOBEC3-enzymes, as suggested by the presence of D67N and E138K in hypermutant and probably defective HIV-DNA strains at T1 of A009. Thus, the presence of these mutations in potentially defected strains might have accelerated their disappearance at following time points .No emergence of new MRMs was observed neither during ATI nor post-ATI, data in line with Clarridge and colleagues, who observed no evidence of emergence of antiretroviral drug resistance mutations within intact HIV proviral-DNA sequences following reinitiation of ART .In line with the disappearance of MRMs observed in APACHE individuals during and after ATI, the analysis of C2-V3-C3 revealed a more pronounced viral evolution in APACHE than in MODAt participants. In particular, a significant C2-V3-C3 divergence was observed from T1 to T3 in the majority of APACHE subjects and in none of MODAt individuals. In parallel with the variation in genetic distance, phylogenetic analyses showed significant differences in the composition of the peripheral reservoir before and after ATI in APACHE individuals, and no substantial differences in MODAt patients. In particular, the pre-ATI HIV-DNA strains did not align closely with the rebound viruses in most of the APACHE participants (5/6). The striking discordance between the pre-ATI sequences and rebound viruses can be explained by a fraction of a potentially large and diverse reservoir capable of reactivating, and probably arisen by different HIV reservoir . These results are in accordance with Salantes and colleagues, that through phylogenetic analyses of HIV-1 env sequences from QVOA and proviral-DNA, showed that pre-ATI reservoir sampling did not predict viral-rebound . DiffereNotably, the apparently different results between Salantes and us can be explained by the distinct populations analysed in the two studies. In our work, the timing of the sampling post-ATI is just at achievement of undetectable VL after ART resumption, and not at least 6 months after viral suppression ,22. ThisDifferently by APACHE individuals, the highly interspersed C2-V3-C3 sequences at the different time-points in MODAt participants support the hypothesis that in presence of continuous viral suppression no substantial changes to the sampled reservoir can occur.Our study has some limitations. We did not evaluate replication-competent virus neither cell-associated HIV-RNA, defined as a biomarker of HIV persistence and latency reversion. The amount and the characterization of viral reservoir in other districts, like lymph nodes, GALT, was not assessed. In order to define viral evolution, we analysed pol and C2-V3-C3 region, and not the whole HIV-1 genome, even if all the analyses provided concordant and reliable results. Due to the conservative criteria we adopted for ART resumption, we could not evaluate the immune-mediated viral control that could theoretically have occurred after an initial transient rebound. Finally, the small number of patients here analyzed limits the power of the study and the possibility to draw general conclusions. Detecting differences between individuals who underwent ATI versus those who successfully continued ART in the considered outcomes, or their determinants remains challenging.In conclusion, our results confirm that a closely monitored short interruption of treatment does not affect the amount of post-ATI peripheral-total HIV-DNA values, nor the efficacy of ART in inducing viral suppression, thus representing a safe intervention. In some patients, it might cause a genetic diversification of peripheral viral reservoir both in term of MRMs rearrangement and in term of viral evolution, that again do not affect the efficacy of antiretroviral drugs used. These results do not exclude that rebound viral strains released from different anatomical HIV reservoirs  might participate in the modification of new peripheral viral composition of post-ATI reservoir. In two cases ATI did not cause peripheral virus rearrangement. Both cases presented reduced viral divergence at T1 as suggested by the limited C2-V3-C3 intra-host variability and the presence of fixed resistant mutations in the almost totality viral population. Thus, we can speculate that the presence of a dominant population at baseline of treatment interruption might be related with lower probability of peripheral reservoir rearrangement during/post-ATI. No patient enrolled in this study achieved long-term virological control in the absence of ART, thus limiting the possibility to draw conclusions about parameters related to HIV control. Thus, a complete definition of key and reliable viral parameters for future HIV-1 cure strategies remains challenging.


Asymptomatic Cryptococcal Antigenemia (CrAg) patients develop meningitis within a month of testing positive. Pre-emptive antifungal therapy can prevent progression to Cryptococcal meningitis (CM). In April 2016, a national CrAg screening program was initiated in 206 high-volume health facilities that provide antiretroviral therapy in Uganda. We report the evaluation of the CrAg screening cascade focusing on linkage to care, fluconazole therapy for 10 weeks and 6 months follow up, and ART initiation in a subset of facilities.We conducted a retrospective, cross-sectional survey of patients with CD4 < 100 at seven urban and seven rural facilities after 1 year of program implementation. We quantified the number of patients who transitioned through the steps of the CrAg screening cascade over six-months follow-up. We defined cascade completion as a pre-emptive fluconazole prescription for the first 10 weeks. We conducted semi-structured interviews with lab personnel and clinic staff to assess functionality of the CrAg screening program. Data was collected using REDCap.We evaluated 359 patient records between April 2016 to March 2017; the majority  were from government owned health facilities and just over half  had a median baseline CD4 cell count of < 50 cell/μL. Overall, CrAg screening had been performed in 255/359  of patients’ records reviewed, with a higher proportion among urban facilities ) than rural facilities ). Among those who were CrAg screened, 56/255  had cryptococcal antigenemia, of whom 47/56  were initiated on pre-emptive therapy with fluconazole and 8/47  of these were still receiving antifungal therapy at 6 months follow up. At least one CNS symptom was present in 70% (39/56) of those with antigenemia. In patients who had started ART, almost 40% initiated ART prior to CrAg screening. Inadequacy of equipment/supplies was reported by 15/26 (58%) of personnel as a program barrier, while 13/26 (50%) reported a need for training about CM and CrAg screening.There was a critical gap in the follow-up of patients after initiation on fluconazole therapy. ART had been initiated in almost 40% of patients prior to CrAg screening.. Higher antigenemia patients presenting with CNS symptoms could be related to late presentation. There is need to address these gaps after a more thorough evaluation. Cryptococcus neoformans is the most common cause of meningitis among adults with Human Immunodeficiency Virus (HIV) in sub-Saharan Africa (SSA), and it is associated with approximately 20–25% of acquired immunodeficiency syndrome (AIDS)-related deaths in this region [s region –4. Despis region , and SSAs region , 9. Amons region , and this region .C. neoformans can be diagnosed using a cryptococcal antigen (CrAg) test in serum/plasma before the infection progresses and the patient develops meningitis. The current management strategy for CM includes regimens that are expensive and not always available, especially in resource-limited settings [Infection with settings , 12, 13.settings –16.The prevalence of asymptomatic cryptococcal antigenemia in people living with HIV in Uganda with CD4 ≤ 100 cells/μl is estimated between 5 and 10% . AsymptoIn order to increase the capacity to detect CM before it progresses to meningitis in patients with HIV in Uganda, a CrAg screening program was initiated in 206 high-volume (≥1000 patients) government and private health facilities providing ART through the Ugandan Ministry of Health.The CrAg screening program was initiated with the training of key members of the treatment teams from US Centers for Disease Control and Prevention (CDC)/U.S. Presidents Emergency Plan for AIDS Relief (PEPFAR) implementing partners. The key members were then tasked to initiate trainings at the facility level. CDC provided the cryptococcal antigen screening kits for use during the roll-out in the 206 chosen health facilities. Since the initiation of the program in April 2016, there has not been a formal evaluation of the operations and outputs of this program. We therefore sought to understand the current operation of the program through; 1) quantification of metrics along the CrAg screening cascade and 2) identification of operational challenges cited by the implementers at selected health facilities.We conducted a cross sectional study with two parts: Part 1 was a retrospective review of patient records in an electronic medical record (EMR) systems (Open MRS)  or in thUsing the initial CrAg kit distribution list from the Infectious Diseases Institute (IDI) directory, we obtained health facilities’ contact information and contacted the selected health facilities via telephone ahead of the planned visit/data collection date. During the call, study personnel explained the study and procedures  and asked about the availability of the laboratory personnel and ART in-charges and the best days/times to schedule a study visit. Study personnel called the health facility approximately 2 days before the planned visit to remind them of the upcoming visit and what to expect during the visit. This was another opportunity for the facility to ask questions about the study. After agreement on the day and time, two study team members then went to the health facilities, did formal introductions, and identified the contact person managing the EMR or CD4 registers.Our evaluation included 14 health facilities located in two urban districts in Central Uganda: Kampala  and Wakiso, and three rural districts in South Western Uganda: Masaka, Rakai, and Ssembabule. The 14 health facilities  were at different tiers of the health care system delivery as follows: (1) level III ; (2) level IV ; (3) general hospitals ; and (4) regional referral hospitals . Health facilities were selected using a convivence sample of those that responded to our request to visit.Using unpublished sample data from 15 HIV programs implementing partners, we estimated the proportion of patients with CD4 count less than 100 cells/μL and screened for CrAg to be 19%. Using a 95% confidence level and an expected error margin of 5%, we expected to review 236 patient charts from each of the two regions, giving an overall sample size of 472 patient charts. We conducted 26 individual interviews out of the 28 planned, targeting laboratory personnel and in-charge of the ART clinic, two from each of the participating 14 selected sites. The two individual interviews could not be conducted due to concurrent events at one of the facility. With a total of 26 interviews, the number was sufficient to reach saturation of the major themes related to the barriers and facilitators for CrAg screening.We abstracted data from the OpenMRS® medical records system or the CD4 and CrAg registers. These registers are standard Uganda Ministry of Health (MoH) tools that are used for documentation and generation of routine performance reports on selected CD4, viral load, and CrAg screening indicators from point of diagnosis to point of treatment or pre-emptive fluconazole (CrAg screening cascade). We operationally defined a successful cryptococcal care cascade when: at least 90% of patients with a CD4 ≤ 100 get a CrAg test, 90% of CrAg positive patients start fluconazole, and 90% of these complete 6 months of fluconazole . We obtathese are patients with HIV who have freely disclosed their HIV status and perform low level tasks at HIV clinics including treatment education, file retrieval and taking patients’ vitals), and the study team abstracted data related to the CrAg screening program. Figure Study personnel, together with the EMR/ CD4 register focal person, generated a list of patient IDs with baseline CD4 ≤ 100 and tracked these in the CrAg/laboratory register to ascertain whether or not they underwent CrAg screening. When information was not available or missing from the CrAg/laboratory register, patient charts were retrieved by peer patients/ expert clients  health facility’s capacity to do CD4 testing for all newly diagnosed HIV patients, 2) staff training on CrAg screening since Jan 2016, 3) stocks of CrAg screening kits and the mechanism for replenishment, 4) capacity (availability of human resource and equipment) of the health facility to do lumbar punctures and 5) availability and prescriptions of fluconazole.On the same day of the CrAg register/chart review or the next day, the study team located the laboratory personnel and ART in-charge and administered the interviews after obtaining their consent. All the data from the registers and interviews were directly entered into a REDCap®  databaseWe developed data abstraction tools that included variables reflecting the different steps along the CrAg screening cascade. The study tools were pre-tested at the IDI HIV clinic within Mulago National Referral Hospital in Kampala, Uganda. We then designed a database and survey using REDCap electronic data capture, hosted by the University of Minnesota , 23. We Our analysis included 359 76.1%) out of the expected 472 patient charts. All but one health facility were government health facilities, and the majority  were health centres at level III (Table % out of P < 0.001). Ninety four percent (210/255) of the CrAg tests were done within 7 days of receiving a CD4 count result. Twenty two percent  of patients screened were CrAg positive . Of the CrAg positive patients, 50/56  presented with at least one CNS sign or symptom and 39/56  had a lumbar puncture performed. While 39/48  of patients with CNS symptoms in urban health facilities had diagnostic lumbar punctures done, none was done in any of the rural health facilities. In patients with confirmed CrAg antigenemia, 47/56  were initiated on fluconazolepre-emptive therapy. At 6 months follow up, 8/47  were still receiving fluconazole pre-emptive therapy. Among patients who had started ART, nearly 40%  started ART prior to having a CrAg test.Of the 359 patients, the overall median (IQR) CD4 cell count was 45 cells/μL 21–71), 193 (53.8%) patients had a baseline CD4 cell count of < 50 cells/μL and 178/359 (49.5%) patients were classified with WHO clinical stage 3 or 4 HIV disease. As illustrated in Tables 1–71, 193Results of semi-structured interviews of the laboratory personnel and ART clinic in-charges are shown in Table In this study, we observed successes as well as critical gaps along the CrAg screening cascade in patients with lower CD4 cell count. Among eligible patients for CrAg screening, seven out of every ten were subsequently screened, over 80% of eligible patients was initiated on fluconazole, pre-emptive therapy but less than one in five of these were still receiving fluconazole after 6 months, in contrast to the Uganda Ministry of Health guidelines, which recommend 6 months of fluconazole pre-emptive therapy for cryptococcal antigenemia. Lack of supplies and training gaps were identified by health workers as impediments to a successful CrAg screening program.Knowing the estimates of opportunistic infections among people living with HIV is very crucial in designing prevention strategies and major treatment needs , this haOur evaluation study found higher CrAg screening in the urban facilities (81.3%) compared to rural facilities (56.7%), this may be associated with decreased logistical supplies in the rural areas, In addition, current WHO guideline recommendations of the ART test and treat policy in resource limited settings with decreased access to CD4 machines so as to prevent further delay in ART initiation, which is so profound in the rural areas.We found a cryptococcal antigenemia prevalence of 22%. This was higher as compared to earlier analyses by Rajasingham et al, which found a global cryptococcal prevalence of 6%  . In addiDespite the fact that a big percentage of patients were initiated on pre-emptive fluconazole after having a positive CrAg, which could be related to the fact that fluconazole is provided free of cost at most of government ART care centres in Uganda, the 6 month follow up was significantly low with only 17.0% still in care and on fluconazole. As demonstrated in number of studies, clinical cryptococcal meningitis develops in a certain number of patients even with pre-emptive fluconazole therapy. A study by Wake et al also showed that patients with cryptococcal antigenemia had 3.3 times increased risk of dying as compared to CrAg negative, and this remained significant even when adjusted for baseline CD4 (< 50 cells/μL) . FurtherWe were unable to locate and review some of the patients’ files and thus our findings are based on a reduced (76.1% of expected patients’ records) sample size. Such lost files were for patients classified as dead at the clinic or lost to follow up, and this arose out of a failure or complete absence of streamlined filing system at the health facility. The failure to review such patients’ files could have resulted into a possibility of under estimating the metrics along the CrAg screening cascade as presented here, this may be avoided in future studies by accounting for higher percentage of samples with missing data in the initial sample size estimation. Secondly, like any other retrospective study, we encountered a number of files with missing data despite the fact that such patients had met the inclusion criteria. Others were withdrawn from the analysis because of poor documentation that made ascertainment of metrics along the CrAg cascade difficult; and this further impacted on our sample size. The passive nature of surveillance in almost all health facilities made it impossible for us to quantify all the patients that had died during the 6 months follow up as some had passed away in the community with no data captured in the patient chart at the facility. Due to lack of a centralised system for HIV care and CrAg screening, there is a possibility that some of the patients referred from lower level facilities to higher level facilities could be counted twice resulting in overestimation of metrics in the CrAg screening care cascade Facilities were selected via a convenience sample, which makes the generalizability of these results limited.There is a critical gap in follow up of patients after they initiate pre-emptive fluconazole therapy. A high proportion of patients were ART experienced and presented with advanced HIV disease, thus exposing them to an increased risk of unmasking cryptococcal meningitis. There is great need for logistical support, test kits and refresher training of health worker personnel on CrAg screening, cryptococcal meningitis management, and lumbar puncture procedures especially in rural facilities. A more thorough evaluation to identify gaps and successes of the CrAg screening program is /warranted.