A complex and negatively reinforcing relationship exists between infection with Human Immune Deficiency Virus (HIV) and malnutrition. HIV-induced immune impairment and its resulting opportunistic infections (OIs) can lead to malnutrition and nutritional deficits, can, in turn, hasten the progression of HIV infection and reduce chances of survival. The determinants of undernutrition among patients receiving antiretroviral therapy (ART) is poorly understood in Ethiopia, despite a high prevalence of food-insecurity that overlaps with a generalized HIV/AIDS epidemic. Therefore, this study aimed to assess determinants of undernutrition among adult patients receiving antiretroviral therapy at Debre Markos Referral Hospital in Northwest Ethiopia.We conducted an institution-based, unmatched, case-control study with 636 adult patients receiving antiretroviral therapy. We randomly selected 212 patients with poor nutritional outcomes (cases) and 424 without undernutrition (controls) and then conducted a chart review to collect information on their treatment, socio-economic, and demographic background. Data were analyzed using bivariable and multivariable logistic regression to identify factors associated with under nutrition.We found that greater age , fair or poor adherence , and the presence of OIs , anemia , or eating problems , were all independently and positively associated with under nutrition. Starting treatment with a medium or low CD4 count was protective . Having social support , and having a source of informal care-giving , reduced the odds of undernutrition.Our findings support calls for treating HIV infection early and aggressively, while closely monitoring patients for opportunistic infections that might affect eating and drug side effects that may affect appetite. The role of disclosure, peer-caregivers and age in preventing undernutrition should be explored in future research. A complex and negatively reinforcing relationship exists between infection with Human Immune Deficiency Virus (HIV) and malnutrition. HIV-induced immune impairment and its resulting opportunistic infections (OIs) can decrease food intake; increase energy expenditure; cause nutrient malabsorption; and alter the body’s ability to utilize and excrete nutrients leading to nutritional deficiencies and poor overall nutritional status . NutritiAntiretroviral therapy (ART) for HIV-infected patients was thought to be a solution to HIV-associated wasting and malnutrition. It is well established that a majority of clinically malnourished people living with HIV who initiate ART will increase or stabilize their weight, even without therapeutic or supplementary feeding support . HoweverContinued study of the relationship between malnutrition and ART is particularly important in low-income countries such as Ethiopia where high levels of food insecurity and poverty overlap with generalized HIV epidemics , 18, 19.Assuring adequate access food in HIV treatment programs has been a central demand of people living with HIV (PLHIV) in low-income countries and, as such, nutritional counseling and therapeutic feeding have been incorporated into HIV treatment programs and tested for efficacy –30.However, our understanding of how undernutrition and HIV treatment interact, and specifically what factors influence individuals’ risk of malnutrition while on ART, for sub-Saharan African counties, is still spotty with a relatively few studies conducted given the large overlap between food insecurity, malnutrition, and HIV-infection in many of these countries. Only a handful of studies on this topic have been conducted in Ethiopia.This study adds to the knowledge base on this topic by assessing the determinants of undernutrition among adult patients receiving antiretroviral therapy at Debre Markos Referral Hospital, a large tertiary hospital in Ethiopia with an established HIV treatment program.This study was conducted at Debre Markos Referral Hospital, in Debre Markos Town, which is located 299 km northwest of the capital city, Addis Ababa. The hospital has a single ART clinic that serves more than 8000 pre-ART and ART clients. The clinic’s patient flow is approximately 1000 monthly with approximately 30 patients daily. At the time of the study, there were 8995 total registered patients at the clinic, of which 3643 were on ART. Of the registered ART patients; 150 were pregnant 38 had been lost to follow up, 271 had died, and 1152 had not been in touch with the clinic for more than three months. The remaining 2022 patients were receiving ART and eligible for the study. Of these, 1700 were over the age of 18 . This wa2. Controls were patients with normal nutritional status (BMI between 18.5 Kg/m2 and 24.9 kg/m2) in 2015. Patients whose patient cards had incomplete data, pregnant women, and patients who had been transferred from other health institutions were excluded from the study for the sake of data quality.This is an institution-based, unmatched, retrospective case-control study. All adult patients on ART who were registered at the Debre Markos Referral Hospital ART clinic between September 11, 2008 to August 11, 2015, and who had complete registration cards showing current follow-up were eligible for inclusion. Cases were patients who had a record of undernutrition in 2015. We define undernutrition as having a BMI < 18.5 kg/mWe calculated our sample size with EpiInfo (version 7), using double-population proportion formula. Our outcome of interest was undernutrition at any endpoint specific for each patient during the study period. We estimated the proportion of the patient population that would be malnourished by reviewing patient records to find how many patients had reported problems with eating, as eating problems was found to be the main determinant of undernutrition in a study population comparable to ours at Gondar Referral Hospital in Ethiopia . In thatWe selected our sample by extracting a list of all of the eligible patients on ART in the clinic from the clinic registration book. This list was divided into cases and controls based on the patient BMIs recorded in the registration book. We then used simple random sampling to select a random sample of participants from the case and control lists using SPSS (version 20).2 and 0 if the patient had a BMI between 18.5 and 24.9 kg/m2 based on the World Health Organization’s (WHO) 2006 BMI classification scale [The dependent variable of the study is undernutrition at any endpoint specific for each patient during the study period. This a dichotomous variable, coded as 1 if the patient had a BMI < 18.5 kg/mon scale .We collected and reported four categories of independent variables  and type of infection; whether the patient had been anemic (measured by hemoglobin level); whether the patient had experienced eating problems or received therapeutic food and the cause of eating problems; the patient’s baseline CD4 count; the patient’s current CD4 count at the time of the study; ART adherence; and the total duration of pre-ART and ART follow up. Patients were said to have had an opportunistic infection, if they developed at least one of opportunistic infections listed in Harrison’s Medicine .The adherence to ART was assessed using percentages and was calculated using number of doses missed for the last one month. Adherence percentage equal to or greater than 95% or ≤ 3 doses missed per month is considered as Good, 85–94% or 4–8 doses missed per month considered as Fair, and less than 85% or ≥ 9 doses missed per month is considered as Poor . FinallyOur main data source was the ART clinic registration book and the patient’s registration card. We checked the completeness of the data in the ART clinic’s registration book before beginning data extraction. We developed a data extraction checklist based on the variables we wanted to include in the study and conducted a pre-test on random sample of patient registration cards , to ensure its consistency and completeness.The data collection process was overseen by the project’s principal investigator. We recruited a team of two public health professionals and a BSc nurse who were not associated with the ART clinic as data collectors. They were given a one-day training on the objectives of the study, the method of data collection and how to extract information from patient registration cards and the registration book using the checklist. We also communicated with individuals from Hospital card office and briefly trained them about the study process and the requirements., their medical records) using the patient’s registration codes. Each patient/registration card was then assigned a unique anonymous identification number, which was recorded on our data extraction checklist. We extracted data from patient registration cards and from the registration book, using the pre-tested checklist. The collected data were entered in EpiData (version 3.1) to minimize data entry error and then exported to STATA (version 14) for analysis.Hospital health informatics technicians assisted us in tracing clinic patients’ hospital registration cards . The study sample was disproportionately female  and most study subjects were between the ages of 18–35 (see Table The sample was predominantly urban (82% of cases and 87% of controls) and relatively well-educated: 32% had a secondary school education or higher. And, reflecting their urban residence, the majority of the sample had access to public municipal services (71% of cases and 80% of controls) and a large proportion, relative to the Ethiopian average, had access to piped water (69% of cases and 54% of controls), electricity (64% of cases and 78% of controls), and a phone (45% of cases and 61% of controls). Almost all respondents (> 98%) owned their own home (Table The average amount of time that patients had been receiving care at the clinic was 5.7 years (±2.1SD) for cases and 5.6 years (±2.4SD) for controls. Most patients started ART with advanced disease: 67% of cases and 51% of controls had a baseline CD4 count < 200 cells/ul before starting ART; and 72% of cases and 57% of controls started treatment at WHO stage 3 of disease or higher. Our sample of patients had been receiving ART for 5.2 years (±1.9SD) for cases and 4.7(±2.1SD) years for controls.Treatment was successful for the majority of patients. Most were rated as having good adherence (76% of cases and 93% of controls), and, at their last follow up visit, most had CD4 counts above 350 cells/ul . In addition, the vast majority was at WHO stage 1 at their last follow up visit (99% of cases and 98% of controls)  while under care (58% of cases and 34% of controls). The most common OIs were tuberculosis, which was experienced by 19% of patients, and which constituted 45% of OIs reported (see Table Sixty-five percent of cases and 26% of controls were recorded as having had a problem with eating during their care but only 10% of patients had received therapeutic food supplementation (28% of cases and 1% of controls). The most common reasons given for having an eating problem were loss of appetite (reported by 55% of those with eating problems), oral candidiasis (53% of patients), and nausea (33.33 of patients)  with spouses, priests and male siblings being the most frequently mentioned  and those with fair adherence had more than double the odds . The other clinical factor strongly associated with developing undernutrition was having experienced an eating problem while on treatment. The odds of becoming malnourished while on ART was 3.40 times higher for patients who had eating problems than those who did not after controlling for demographic factors, socio-economic support, adherence and for baseline severity of disease . Having higher baseline CD4 counts reduced the odds of developing undernutrition  for CD4 counts 200–350 cells/ul, and  for CD4 counts > 350 cells/ul).The presence of opportunistic infections was also significantly associated with undernutrition after controlling for other factors; patients who had developed OIs while on treatment had 70% higher odds of developing undernutrition, holding other factors constant, compared with those who had not had OIs . Patients who developed anemia had 1.81 times the odds of developing undernutrition than those who did not .Turning towards social and economic support factors, we find that disclosure of HIV status and having a source of informal care-giving support reduced the odds of developing undernutrition  and AOR = 0.48, 95% CI: 0.27, 0.84) respectively. Owning a phone halve the odds of developing undernutrition in bivariable regression. The significance and magnitude of the effect fades in multivariable analysis but approaches significance at the 10% level .We examined the data to see if our strongest associations were modified by gender or educational status. We found no evidence that the associations between undernutrition, eating problems, adherence, disclosure, or having a caregiver differed for women, the more highly educated, or urban dwellers Table .This study set out to examine the factors associated with adult ART patients developing under nutrition during the course of their treatment. We found that greater age, poor adherence, starting treatment with advanced disease and the presence of OIs, anemia, or eating problems, were all independently and positively associated with undernutrition. Having social support and having a source of informal care-giving, reduced the odds of undernutrition.Many of these findings are unsurprising and in line with those recent studies on this topic. It is, for example, logical that the patients with OIs and eating problems would have dietary deficiencies and problems absorbing nutrients and that this, in turn, would lead to anemia and malnutrition. The link between low BMI and anemia among HIV-infected patients is well established  and EthiHowever, expected these results are, they underscore the importance of nutritional counseling focused on loss of appetite and nausea, close monitoring of anemia, and aggressive treatment of opportunistic infections, particularly those that can cause eating problems such as oral hairy leukoplakia, and oral and esophageal candidiasis. Of the OIs, tuberculosis was the most strongly associated with undernutrition and should be targeted for close monitoring.Therapeutic feeding—“food by prescription”—has been shown to be effective at preventing and treating malnutrition in HIV-infected adults  and at iThe strong association that we found between ART adherence and undernutrition is also fairly well established , 15, 24 Research on the association between adherence and nutritional status, suggest a circular relationship, in which poor baseline nutritional status makes ART initiation and adherence more difficult and poor adherence, because it harms viral suppression, leads to under nutrition , 14. In In this study, having disclosed one’s HIV status and having someone to count on as an informal caregiver reduced the odds of becoming malnourished. Previous research has found that disclosure and family support are associated with better prevention and treatment outcomes –50 and wWhen we look at disclosure and nutritional outcomes specifically, the association is not as well established. Most studies examining nutritional outcomes do not include disclosure as an explanatory variable; and the studies of nutritional outcomes in ART patients in Ethiopia that did include it find no association . Given tOur finding patients without a caregiver were two times more likely to develop undernutrition compared with patients with caregivers is also relatively novel as this variable is not included in most nutritional status. Even though research is limited on this topic, our findings are not surprising as it is intuitive that having an informal caregiver would help people with living HIV to adhere to treatment and to maintain their quality of life. Given the strong association found here, further study of the role that caregivers can play in nutritional counseling and support may be warranted. In addition, when we look at sources of support, we find that the “other” source, which we believe would most likely be a community volunteer or a friend, was the most strongly associated with good nutritional status. Similarly, disclosure to a friend was more strongly associated with good nutritional status than disclosure to family members or priests. This suggests that HIV groups and community-based care programs may be an important source of information and support and should continue to be developed.A result that is surprising is the lack of association found between our nutritional outcomes, and our proxies for socio-economic status. We found no association between undernutrition, educational level, urban residence, home ownership and access to public municipal goods such as electricity and piped water. Wealth or income of patients on ART has been found to be negatively associated with undernutrition in Haiti , and BraFinally, our study found that age and poor nutrition status were positively related. The evidence on age as a risk factor for undernutrition is mixed. It was found to have weak positive association with malnutrition in Brazil, no effect on low BMI in Haiti, and a negative association with malnutrition in Senegal. In older HIV-infected adults, more advanced age is associated with lower BMI  althoughThis study has several limitations. First, it is a single-facility study that include only patients that are currently in care, which limits its generalizability. Second, it uses existing patient records its secondary data source, which limits the number of variables that we can include in our models. For example, we were not able to include information on household dietary diversity, baseline BMI and food security, or patient occupation, all of which might have had important associations with nutritional status. Most important, because our outcome variable measured whether the patient had ever developed undernutrition while on treatment, we were not able to determine the direction of relationships between some patient clinical characteristics such as adherence or having had an OI and developing undernutrition.Our findings suggest that ART clinics like those at Debre Markos Referral hospital’s and other similar settings of the country should develop tools and procedures for the early diagnosis of eating problems, anemia and opportunistic disease so that they can intervene in a timely manner during patient follow-up visits. Treatment should be started as early as possible with adherence support and close monitoring of patients for opportunistic infections that might affect eating and drug side effects that may affect appetite. The role of disclosure, peer-caregivers and age in preventing under nutrition should be explored in future research. Support group and community/home-based care programs should be explored for patients living with HIV should be encouraged as a means for encouraging disclosure, providing support, and sharing strategies for maintaining a good nutritional status.


It is not completely clear whether a very high pre-therapy viral load (≥ 500 000 copies/ml) can impair the virological response. The aim of this study was to examine the influence of very high baseline HIV-RNA levels on long-term virological responses under one type of regimen.A retrospective study was performed based on data from two multicenter cohorts in China from January to November 2009, and from May 2013 to December 2015. Untreated HIV infected adults between 18 and 65 years old were recruited before receiving non-nucleoside reverse transcriptase inhibitor-based regimen. All patients had baseline HIV-RNA levels over 500 copies/ml, good adherence, and were followed for at least 24 weeks. Virological suppression was defined as the first HIV-RNA < 50 copies/ml. Virological failure was defined as any of incomplete viral suppression  and viral rebound . Chi-square test, Kaplan–Meier analysis, Cox proportional hazards model and Logistic regression were used to compare virological response between each pretreated viral load stratum.P < 0.001). Very high baseline HIV viremia over 500 000 copies/ml were found to be associated with delayed virological suppression  as well as incomplete viral suppression  and viral rebound .A total of 758 treatment-naïve HIV patients in China were enlisted. Median follow-up time (IQR) was 144 (108–276) weeks. By week 48, rates of virological suppression in three groups  were 94.1, 85.0, and 63.8%, respectively (Very high levels of pre-treatment HIV-RNA were related with delayed efficacy of NNRTI-based ART and increased risk of treatment failure. More potent initial regimens should be considered for those with this clinical character. As the primary treatment for HIV infected patients, the benefits of highly active antiretroviral therapy (ART) in reducing mortalities by suppressing the plasma viral load to an undetectable level has been well established –3. HowevVirological response to ART could be related to several factors including age, sex, mode of transmission, drug adherence, drug resistance, hepatitis virus coinfection, and pretreatment HIV-RNA levels . Among tThe combination of two nucleoside reverse transcriptase inhibitors (NRTI) + non-nucleoside reverse transcriptase inhibitor (NNRTI) has been recommended as the first-line treatment until 2015, when integrase strand transfer inhibitor (INSTI) based regimen were established . HoweverA retrospective study was performed based on two completed prospective multi-center cohort in China. Details of both cohorts have been described in detail elsewhere , 15. The3, 100–199 cells/mm3, 200–350 cells/mm3 and >  350 cells/mm3. Plasma HIV-RNA level and CD4 cell count at each follow-up after the start of treatment  were also collected for analysis.Information including age of diagnosis, sex, mode of transmission and clinical characteristics such as HIV subtype, baseline HIV-RNA level, baseline CD4+ T cell count, planned regimen, year of ART initiation and co-infections including seropositivities of hepatitis B virus surface antigen (HBsAg) and hepatitis C virus antibody (HCV-Ab) were collected. Pre-treatment HIV-RNA levels were categorized as < 100 000 copies/ml, 100 000 to 500 000 copies/ml and ≥ 500 000 copies/ml. Baseline CD4 cell count were categorized as < 100 cells/mmFor the available pre-treatment resistance data, we used the Genotypic Resistance Interpretation Algorithm – HIVdb Programme  to calculate penalty scores for relevant NRTI and NNRTI, and sequences were determined to be either susceptible  or resistant .The prospective cohorts in this study had been approved by Institutional Review Board of Peking Union Medical College Hospital and complied the Principles of Good Clinical Practice and the Declaration of Helsinki. Patients were enrolled in the cohorts after informed consents were signed to provide their anonymized data for academic not-for-profit studies.Definitions of virological suppression and virological failure differ between different regions , 17. TheCI). Patients were right-censored if they did not achieve virological suppression but stopped follow-up. Binary Logistic regression was used to evaluate the odds ratio (OR) to incomplete viral suppression and viral rebound. Factors with associations with P <  0.10 in univariate analysis were entered into the multivariable model. All tests of significance were 2-sided, with a P value < 0.05 indicating that an association was statistically significant.All statistical analyses were performed using the SPSS 24.0 statistical software package  and GraphPad Prism version 8 . Baseline demographic and clinical characteristics were summarized using medians (interquartile ranges [IQRs]), and frequencies (percentages). Chi-squared test was used in analyzing virological suppression rate in three different groups. Kaplan-Meier curves were used to estimate the time and probability to achieve virological suppression. Multivariable Cox proportional hazard models were used to adjust for potential confounders and outcomes were expressed as ratio hazard (RH) with confidence intervals of 95% confidence interval (95% 3. The most commonly used regimen was tenofovir disoproxil fumarate (TDF) + lamivudine (3TC) + efavirenz (EFV) (517 [68.2%]). In those whose genetic testing were available, very few had significant resistance to their regimen (16/353 [4.5%]). The median follow-up time of the study population was 144 (108–276) weeks.The study sample was based on 758 eligible pre-treatment HIV infected patients. Demographic and clinical characteristics of the included patients are shown in Table P < 0.001). In addition, the cumulative Kaplan-Meier estimation showed a significantly lower probability of virological suppression for higher HIV-RNA categories  cells/m3 at week 96. Table Overall, 529 (69.8%) of the patients in the cohort had achieved virological suppression by week 24, and 671 (89.7%) patients achieved virological suppression by week 48. At week 24, only 18/48 (37.5%) patients with a baseline HIV-RNA over 500 000 copies/ml had achieved virological suppression, compared with 117/211(55.5%) in those with a baseline between 100 000 and 500 000 copies/ml. Figure CI: 0.319–0.648; P <  0.001). Male sex was shown to be another significant relevant factor of deterred virological suppression . None of age, pre-treatment CD4 cell count, mode of transmission, HIV-subtype, pre-treatment resistance, initial regimen, sero-positivity of HBsAg of HCV-Ab was significantly associated with delayed virological suppression.Univariate and Multivariable Cox proportional hazard models were used to analyze factors associating with time to virological suppression Table . Very hiIn the sample cohort, 71/758 (9.4%) patients had incomplete viral suppression, among whom 21/71 (30.0%) had never achieved virological suppression. 23/758 (3.0%) patients had viral rebound, with 7/23 (30.4%) having additional blips, 1/23 (4.3%) having severe adverse effect and stopped the initial regimen in week 48. Of these patients, 3/94 (3.2%) were resistant to their initial regimen that was later proved by genetic analysis.OR) to incomplete viral suppression were significantly higher in patients with baseline HIV-RNA levels ≥ 500 000 copies/ml . After excluding patients with viral blips, baseline HIV viremia over 500 000 copies/ml were found to be independently associated with rebound .Binary Logistic regression models were built to evaluate factors related to incomplete viral suppression or viral rebound Tables  and 4. AAs reported by many previous studies, including some large-scale studies with over 100 000 participants, that baseline HIV viral load over 100 000 copies/ml is associated with longer time to virological suppression as well as increased incidence of viral rebound –8, yet vP = 0.896).INSTI-based ART, in specific dolutegravir (DTG)- or raltegravir (RAL)-based regimens have been recommended as the first-line regimen in most developed countries , 17 for Recent guidelines have recommended starting ART as soon as HIV infection is confirmed, regardless of HIV-RNA level or time of infection so as to get rapid suppression of viral load . In the P = 0.136). In sub-analysis within each HIV-RNA group, male sex was still significantly related with postponed virological suppression . The association between male sex and postponed virological suppression in this study is consistent with the result of one previous study [In the present study, male sex was found to be an independent risk factor of delayed virologic suppression in multivariate Cox proportional hazard analysis but not significantly related to incomplete suppression or viral rebound. We made an additional comparison of proportions of male and female patients in each viral load stratum, finding that there was no difference in distribution of both sexes  patients. In this analysis, however, the OR of virological failure increased as we raised the viral load stratum. The result would be more convincing if more cases were included. Secondly, adherence in this study was assessed by self-report, which may be less accurate than other methods such as pharmacy refilling, electronic adherence monitoring device [The present study has some limitations. Firstly, the 95% g device . As the g device . We analg device , yet theOur findings in this study support that very high pre-treatment HIV-RNA levels are associated with impaired virological response. Male sex was also found to be related to postponed virological undetectability. Patients with very high HIV-RNA levels, especially those with baseline RNA over 500 000 copies/ml may possibly need some potent regimens as their initial treatment.Additional file 1: Table 1S. Number of patients that achieved virological suppression and number of patients that remained in the cohort in each follow-up checkpoint.


In resource poor countries like Ethiopia, little is known about the survival of patients treated with antiretroviral therapy which depends on different factors. Evidence shows that mortality has been high particularly in the first 3 to 6 months of initiating antiretroviral therapy. Hence, the study aimed to assess the Epidemiology of survival pattern and its determinants among adult HIV positive patients on highly active antiretroviral therapy.Retrospective cohort study was employed among a total of 455 records of patients who were enrolled on antiretroviral therapy from September 2006 to August 2010. Socio-demographic, clinical, immunological, behavioral, and date of antiretroviral treatment initiation including date of follow up status were extracted. Significant predictor variables were identified by fitting Cox’s proportional hazard model using a backward stepwise method and statistical significance variables were declared based on a p-value less than 0.05.A total of 455 adult HIV/AIDS patients on ART contributed to 886.05-person-year of observation and 65.7% were alive and on treatment, 17.1% were lost to follow up and 7.5% died. The study showed that the estimated mortality was 4.4%, 5.3%, 6.1%, 7%, 7.5% and 7.5% at 6, 12, 24, 36, 48 and 60 months of follow up period, respectively. The overall incidence rate of mortality was 4.2 per 100 person-years of observation. In multivariate analysis age 45 and above , bedridden functional status , poor ART drug adherence , Tuberculosis co-infection , non-disclosure  and severe anemia  were found predictors.Patients with older age, tuberculosis infection, bedridden patients and severe anemia were predictors. Tracing poorly adhered patients and giving drug counseling as well as encouraging them for disclosure to their families is crucial to improve their survival. Survival time for infected Human immune virus (HIV) patients is the survival of HIV patients from the date of initiation of antiretroviral therapy (ART) to either death, loss to follow-up, transfer to other health institutions or to live follow-up . GloballIn developing countries especially in sub-Saharan Africa, through further decentralization, treatment coverage can hopefully continue to improve patients in care. However, due to co-infections, early mortality remains high, especially among individuals with advanced diseases , 4. EvidEven though access to ARV therapy has shown significant clinical improvement by achieving the goal of therapy, many early deaths are associated with socio-demographic, behavioral risk and health factors –8. Two yEthiopia has adopted a test-and-treat offering ART to all PLHIVs regardless of CD4 count, enabling more PLHIVs to have access to ART that can further reduce morbidity and mortality as well as the number of new infections. Approximately 610,000 people are living with HIV in Ethiopia in 2017, with approximately 15,000 deaths/year , 4, 12. Although there has been an increasing interest among researchers in determining factors that influence the survival of people in HAART, there is no evidence available to identify determinants of mortality in patients receiving ART care and support services in public health facilities in the study area. The aim of this study was therefore to assess the Epidemiology of survival pattern and its determinants among adult HIV-positive patients on highly active antiretroviral therapy in public health facilities in Sawla Town, Southern Ethiopia.A retrospective cohort study was conducted from March 01, 2019, to April 10, 2019, in two public health facilities for patients who were started ART from September 2013 to August 2018. These health facilities are Sawla General Hospital and Sawla Health Center located in Goffa Zone, Southern Nation Nationalities People Regional State in southern Ethiopia. The town is located 285 km from Hawassa and 515 km from Addis Ababa, the capital of Ethiopia, in Southern direction. These two health facilities serve more than 1 million people from the surrounding. Since the establishment of Hospital (2002) and Health center (1998), a total of 920 (394 on treatment during 2011) patients started ART in Hospital and a total of 1094 (519 on treatment during 2011) patients started ART in Health center.The study population consisted of all randomly selected adult HIV positive individuals (15 years of age and above) who started HAART between September 2013 to August 2018 in Sawla General Hospital and Sawla health center. HIV positive adults aged 15 and above who started ART with complete baseline data and intake form were included in the study. But, HIV-positive individual records having incomplete baseline data were excluded from the analysis. Among 467 electronic patient records reviewed, 308 were alive, 80 lost, 45 transferred out and 34 died. Totally, 8 with incomplete electronic records  and 4 patients who started ART since August 2010 were excluded from the study. The sample size was calculated by using sample size estimate for Longitudinal Studies with 95% confidence level, 80% power, one exposed to one non-exposed allocation ratio and the event of interest in exposed  and non-exposed group  . Then, tHAART-initiated patients were followed until the date of death, loss to follow-up, transferring out, or completion of the study. Individuals who were on HAART, lost to follow-up, or had transferred out at the end of the study period were censored; that is, they were considered to be alive for the time that they had been under follow-up. The outcome event studied was survival and that censoring occurred for loss to follow up, transfer or study completion. The survival time was calculated in months using the time between the dates of treatment initiation and the date of the event (death) or date of censoring. Drug adherence was assessed in the study patient medication diary from the record. Then classification was done based on WHO classification and poor adherence was defined if the percentage of the missed dose was between < 85% (> 6 doses of 30 doses or > 9 doses of 60 doses) as documented by ART physician, fair adherence was defined as if the percentage of the missed dose was between 85 and 94% (3–5 doses of 30 doses or 3–9 dose of 60 doses) as documented by ART physician, and Good Adherence was defined as if the percentage of missed dose is between > 95% (< 2 doses of 30 doses or < 3 dose of 60 doses) as documented by ART physician. Substance use in this study was referred to as use of at least one of the substances commonly known in Ethiopia . In our study TB co-infection was assessed by taking clinical lab data.The electronic database format which was developed by the Communicable Disease Control (CDC) was used as a data extraction tool. Secondary data routinely collected for clinical monitoring and evaluation purposes in the health facilities were used. Data was extracted retrospectively from all eligible electronic records of adult HIV/AIDS patients on ART from the ART database form. First, the profiles of all patients who started ART from September 2013 to August 2018 were evaluated and patients who started ART since August 2018 were excluded. Then, information about study participants, such as socio-demographic characteristics, clinical and immunological characteristics, behavioral characteristics, medication and prophylaxis, and survey endpoints were retrieved from the clinical records in the electronic database of HIV/AIDS patients by trained ART data clerks. Two ART data clerks who were trained on ART data management were recruited from the Hospital and Health center. In order to ensure the quality of collected data, one-day intensive training was given for data extractors about the objective and techniques of data extraction. After 1 day training, data extraction was started and different documents for the same patients were triangulated in case of odd values, non-logical data, or missed data.STROBE checklist was used to analyze and report data . The ExcA total of 467 HIV-positive individual records were reviewed. Of these, 455 (97.4%) had complete socio-demographic, baseline clinical, and immunological data. Out of 455 patients on HAART, more than half (58.3%) were females and the mean age was 31.5 (SD = 9.4) years. More than half, 60.4%, of the patients lived in urban areas. Nearly half (49.7%) of them were Orthodox Christian by their religion, about two-third (65.6%) of them were married and 38.7% had primary education  respectively. Concerning with preventive prophylaxis, more than three-fourth (78.7%) patients received cotrimoxazole preventive therapy (CPT) and about 77.6% patients received Isoniazid prophylaxis at the time of ART initiation. Out of 455 patients, 67 (14.7) patients had tuberculosis co-infection at the time of ART initiation and majority (41.1%) of them were in WHO stage I at the time of ART initiation. Our finding revealed that majority (84.4%) of them had good ART adherence and most of them (73.4%) initiated ART at CD4 ≥ 200 cells/μL ]. The median hemoglobin level and CD4 counts were 11.6 mg/dL  and 309 cells/mmOut of 455 cohorts of adults ART patients, 299 (64.9%) were alive and continued their treatment in the health facilities, 78 (17.4%) were lost follow up and 34 (7.5%) died. The estimated mortality rate was 4.4%, 5.3%, 6.1%, 7%, 7.5% and 7.5% at 6, 12, 24, 36, 48 and 60 months follow up period, respectively. Out of 34 deaths, 20 (58.8%) died within the first 6 months. The overall incidence rate of mortality during ART treatment was 4.2 per 100 person-year observations (PYOs).Candidate variables that were associated with time to death at 25% significant level in the bivariate Cox regression analysis were sex, age, educational level, baseline weight, baseline BMI, baseline CD4 count, baseline hemoglobin, baseline WHO clinical staging, baseline functional status, baseline evidence of TB, tobacco smoking, alcohol drink, Khat chewing, disclosure status, ART adherence, CPT and INH prophylaxis. Then, candidate variables that were associated with time to death at 25% significant level in the bivariate Cox regression analysis were entered into multivariate Cox regression analysis. Predictor variables in multivariate Cox regression analysis were bedridden functional status, presence of tuberculosis at baseline, non-disclosure status, age 45 and above, severe anemia and poor ART drug adherence were statistically significant for death in HIV patients at P < 0.05 Table .Table 3MThe findings from the registered cohort showed that there were 34 (7.5%) deaths, providing an incidence density of mortality during ART treatment 4.2 per 100 person-year observations which was higher compared to other studies conducted in Ethiopia , 19 but Northwest Ethiopia  and CameThis study showed that the majority of deaths were occurred in the first 6 months of ART initiation which was comparable to previous study . The estPatients with poor ART adherence had the highest risk of death with 4.5 times more likely to die than adherent patients. Similarly study conducted in Ethiopia showed non-adherent participant’s had a mortality of 27 times compare to adherent ART patients . Another3 at baseline. This is consistent with studies in Ethiopia [The mortality rate in this study was associated with baseline tuberculosis co-infection. People who started ART with tuberculosis co-infection were found to die almost 4 times higher than those who did not have baseline tuberculosis co-infection. These are explained by 67 HIV/AIDS patients with tuberculosis infection; 82% were in WHO clinical stage III and IV, 73% were ambulatory and bedridden, and 55% had CD4 counts of less than 100 cells/mmEthiopia , 25 and Ethiopia  which foPatients who had severe anemia at baseline were 5 times higher risk of mortality than those who didn’t have. Similar studies done in Ethiopia , 18, 27 Previous studies reported that the age of the patient was found to be the predictor of mortality, where the majority of patients in older age were more likely to die , 29. SimThis study found that patients with non-disclosure status were at high risk of death compared to patients with disclosure status. This finding is comparable to the study conducted in other parts of Ethiopia . This maA patient who initiated ART as bedridden (inability to attain self-care in the daily living) had the shortest survival rate than working (able to perform routine activities). Patients with bedridden functional status had 17.4 times higher risk of death compared with working patients which is similar with findings from Ethiopia , 30.Although this study has strengths such as cross-checking of electronic database data with different documents at the time of non-logic and incomplete data, mortality may be underestimated due to loss of follow-up in patients likely to include more individuals dying at home without being reported.This study had identified independent significant predictors of survival in patients living with HIV/AIDS after the initiation of HAART. These factors include patients with older age, tuberculosis co-infection, bedridden functional status and severe anemia (hemoglobin less than 7 mg/dl). Hence, careful monitoring of patients, tracing poorly adhered patients and providing drug counseling and encouraging disclosure to their families is crucial to improve their survival. Moreover, special emphasis and closer follow-up should be given for HIV-TB co-infected patients.


As in other sub-Saharan countries, the burden of depression is high among people living with HIV in Malawi. However, the association between depression at ART initiation and two critical outcomes—retention in HIV care and viral suppression—is not well understood. Prior to the launch of an integrated depression treatment program, adult patients were screened for depression at ART initiation at two clinics in Lilongwe, Malawi. We compared retention in HIV care and viral suppression at 6 months between patients with and without depression at ART initiation using tabular comparison and regression models. The prevalence of depression among this population of adults newly initiating ART was 27%. Those with depression had similar HIV care outcomes at 6 months to those without depression. Retention metrics were generally poor for those with and without depression. However, among those completing viral load testing, nearly all achieved viral suppression. Depression at ART initiation was not associated with either retention or viral suppression. Further investigation of the relationship between depression and HIV is needed to understand the ways depression impacts the different aspects of HIV care engagement. The prevalence of HIV in sub-Saharan countries such as Malawi are among the highest in the world , 2. LikeDepression is a major contributor to the burden of disease and disability and is highly prevalent among people living with HIV in Malawi and elsewhere in SSA, a region where mental health care is often limited , 16. DepDepression has been shown to be an important barrier to linkage to care, retention, ART adherence and ultimately long-term viral suppression across the globe , 24–29. This analysis addresses this knowledge gap by generating evidence on the association of depression with two key HIV care outcomes: retention in HIV care and viral suppression. Compared to those without depressive symptoms, we hypothesized that those with elevated depressive symptoms at ART initiation would be less likely to be retained in HIV care and to be virally suppressed 6 months after ART initiation.This study is nested within the first phase of a pilot program that integrated depression screening and treatment into routine HIV primary care using existing staff at two public health clinics in Lilongwe, Malawi. We implemented this program in two staggered phases—a depression screening-only phase and a depression treatment intervention phase . This stDuring the screening-only phase, HIV testing and counseling (HTC) counselors and ART providers screened patients for depression at ART initiation using the Patient Health Questionnaire-9 (PHQ-9). The PHQ-9 is a nine-item questionnaire that assesses the presence and frequency of the nine Diagnostic and Statistical Manual of Mental Disorders symptoms of major depression . It has All non-pregnant adults (18 years or older) newly initiating ART at the study sites were eligible to be enrolled in the program evaluation. Research assistants approached such individuals awaiting ART initiation to invite them to participate in the study and nearly all (96%) provided informed consent. This analysis is restricted to consenting individuals who completed depression screening during the screening-only phase of the program, e.g. only those who initiated care between April 2017 and October 2017 at Clinic A and between April 2017 and March 2018 at Clinic B  and accompanying electronic medical record for the patient where HIV care data will be recorded. At each appointment, the clinicians schedule the next follow-up appointment and provide the patient with a supply of ART that will last until their next appointment. ART pills are provided in increments of 30. Generally, for the first 6 months of care, newly initiating ART patients are scheduled to come monthly, though exceptions can be made and a larger supply of ART can be provided.Elevated depressive symptoms suggesting a high risk of a depressive disorder (hitherto described as “depression”) was defined as a PHQ-9 score ≥ 5 at ART initiation, following the definition in the parent study . We alsoWe defined viral suppression as a viral load < 1000 copies/mL drawn at least 5 and a half months (166 days) after starting ART, following the 2018 Malawian ART guidelines. Viral load testing was performed at Bwaila Hospital in Lilongwe using collected plasma or dried blood spots and processed by Abbott m2000 RealTime HIV-1 assay instruments .We constructed several measures to capture retention in HIV care through 6 months in care. Some measures were constructed specifically for this analysis, while others were constructed in alignment with nationally and internationally recognized measures of retention.Continuous engagement in care was defined as never being more that 14 days late to an appointment through 6 months in care.“Alive and in care” at 6 months was defined as being on ART for at least some portion of the 2 months prior to the 6-month anniversary of starting ART, in alignment with Malawi Ministry of Health reporting practices. According to the Malawi Guidelines for Clinical Management of HIV in Children and Adults, an ART patient is classified as a “defaulter” if they go 2 months without ART based on the pills provided at their most recent appointment . PatientCurrently on ART at 6 months was defined as currently having ART on hand at 6 months, i.e. attending an appointment prior to the 6-month point and receiving a supply of ART that would last through the 6-month point. For example, if a participant attended an appointment 5 and half months after starting ART and received a 30-day supply of ART, they would have been considered “currently on ART” at 6 months. This indicator aligns closely with the PEPFAR definition of “currently on treatment” .In care after 6 months was defined as attending at least one appointment where ART is provided after 6 months in care. This definition aligns more closely with the UNAIDS Global AIDS Monitoring 2019 Indicators by identifying participants known to be on ART at some point 6 months after initiation .HIV appointment attendance was defined as the proportion of scheduled HIV appointments attended within one week of the scheduled appointment date in the first 6 months of care .Consistent ART was defined as never going more than 5 days without ART in the first 6 months, calculated from the cumulative days’ supply of ART dispensed at each appointment in the first 6 months and the time between appointments.ART pill possession ratio was defined as the proportion of the first 6 months (183 days) since ART initiation with ART in hand, calculated from cumulative pills dispensed at each ART appointment through 6 months .A combined outcome of continuous engagement in HIV care with viral suppression 6 months after starting ART was defined as meeting both of the following criteria: never being more than 14 days late to a scheduled HIV appointment through 6 months in care and having a viral load < 1000 copies/mL drawn at least 5.5 months (166 days) after ART initiation.Due to the data collection design of this study, only routinely collected clinical data was captured including age, sex, clinic, World Health Organization (WHO) HIV clinical stages for HIV surveillance , villageWe first completed unadjusted, tabular comparisons of HIV care outcomes by depressive severity . We then used log-binomial models to estimate adjusted risk ratios comparing the probability of each binary HIV care outcome among those with depression compared to those without. For the continuous outcomes, we used ordinary least-squares linear regression models to estimate mean differences. Potential confounders were identified through directed acyclic graph (DAG) analysis.To address missing outcome data for those who transferred to a non-study facility or who returned for care around 6 months but did not get a viral load, we used multiple imputation by chained equations (MICE) to fill missing values using logistic regression imputation methods . AssuminAll analyses were performed using STATA IC 14.The National Health Sciences Research Committee of Malawi (NHSRC) and the Biomedical Institutional Review Board (IRB) of the University of North Carolina at Chapel Hill approved the study protocol. All participants underwent a consent process during ART initiation and gave written informed consent to allow the abstraction of their clinical data. During the consent process, the research assistants explained that participation was voluntary, would not affect care provided at the facility, and would not be necessary to receive ART.Of 1091 participants screened, 602 (55%) screened negative on the PHQ-2; the full PHQ-9 was administered to the remaining 489.  , 37, 57,Finally, complexities in measuring engagement and retention in HIV further complicate the study of the role of depression in HIV care. Despite the importance of retention to successfully treating HIV, there is no recognized “gold standard” measure for retention in care . A recenDue to the implementation science nature of this study, the covariates captured were limited to routinely collected clinical data. It is possible that the presented analyses were biased by unmeasured confounding factors such as stigma, socio-economic status, or transportation barriers. It is also possible that we overestimated viral suppression as viral loads could only be drawn from patients who remained in care and a large proportion of patients who did return for care were never sent for viral load testing.This study documented a high prevalence of depression among patients newly initiating ART and low retention in care at 6 months. However, the examined HIV care outcomes at 6 month were similar between those with and without depression. Further research is needed to understand the mechanisms through which depression may undermine different aspects of the HIV care continuum, from testing through sustained retention and ultimately viral suppression.


Little evidence exists to comprehensively estimate adolescent viral suppression after initiation on antiretroviral therapy in sub-Saharan Africa. This study examines adolescent progression along the HIV care cascade to viral suppression for adolescents initiated on antiretroviral therapy in South Africa.n=1080) by 2015 in a health district of the Eastern Cape, South Africa, were interviewed in 2014–2015. Clinical records were extracted from 52 healthcare facilities through January 2018 (including records in multiple facilities). Mortality and loss to follow-up rates were corrected for transfers. Predictors of progression through the HIV care cascade were tested using sequential multivariable logistic regressions. Predicted probabilities for the effects of significant predictors were estimated by sex and mode of infection.All adolescents ever initiated on antiretroviral therapy (p< 0.001) and who experienced decentralised care  were more likely to have a recent viral load. The average effect of decentralised care on recent viral load was greater for female  and sexually infected adolescents . Of the total cohort, 47.5% were recorded as fully virally suppressed at most recent test. Only 23.2% were recorded as fully virally suppressed within the past 12 months. Younger adolescents  and those on ART for ≥2 years  were more likely to be fully viral suppressed.Corrected mortality and loss to follow-up rates were 3.3 and 16.9%, respectively. Among adolescents with clinical records, 92.3% had ≥1 viral load, but only 51.1% of viral loads were from the past 12 months. Adolescents on ART for ≥2 years (AOR 3.42 [95%CI 2.14–5.47], Viral load recording and viral suppression rates remain low for ART-initiated adolescents in South Africa. Improved outcomes for this population require stronger engagement in care and viral load monitoring.The online version contains supplementary material available at 10.1186/s12879-020-05742-9. The expansion of access to antiretroviral therapy (ART) has enabled more children vertically infected with HIV to survive into adolescence . In paraPrevious studies have demonstrated that, even on treatment, adolescents exhibit the worst health outcomes compared to all other age groups , 5. In SAlthough progress along the HIV care cascade has been well-documented globally for adults and adult key populations, less evidence exists for adolescents living with HIV , 7, 9. Acurrent reality of adolescent HIV care progression to the final cascade step of viral suppression in South Africa, with data that reflects contexts beyond well-resourced clinics or urban centres : Age (younger adolescents aged 10–14 years vs. older adolescents aged 15–19 years) [2]; sex [3]; residential location at baseline  [4]; sexual vs. vertical mode of infection , University of Cape Town , Eastern Cape Departments of Health and Basic Education, and ethical review boards of all participating healthcare facilities. At both study waves, adolescent participants and their caregivers provided voluntary, informed, and written consent for participation, including interviews and access to adolescents’ clinical records. Initial review of patient registers in healthcare facilities was only used to identify eligible participants, and further access to clinical records was obtained only after adolescents consented to study participation. In cases of low literacy among adolescents or caregivers, all information and consent procedures were read aloud in the participant’s preferred language. There were no financial incentives for study participation, but all participants received a certificate of participation, snacks, and a small gift pack including pencils and soap. Adolescents who refused to participate were still given snacks. To minimise risk of stigma, all potentially publicly available materials, such as participation certificates and general information pamphlets, referred to the study as one about general adolescent health in South Africa.n=36) and 16.9% (n=183), respectively. Overall, 37.9% of all facility-designated LTFU patients (n=95) were found to be misclassified: 7.4% (n=7) due to unrecorded mortality, and 30.5% (n=29) from re-entry into care after silent transfer to a new facility.A total of 1080 ART-initiated adolescents were recruited into the study. Years of ART initiation ranged from 2000 to 2017, providing 6921 person-years of study follow-up across participants. Median age at study enrolment was 13 years (IQR: 11–16 years), and 55.3% of participants were female (Table n=951) had at least one clinical record available  of participants having records from at least two facilities. In total, 51.3% (n=488) experienced decentralised care.Of the total adolescent cohort, 88.1% , and median follow-up time since ART initiation was 7.2 years (IQR: 4.7–9.8 years). At baseline on ART, the majority of adolescents were viraemic, with 72.1% reporting detectable viral loads (≥50 copies/mL)  had at least one viral load recorded within their files  and 51.1% (n=449) had their most recent viral load recorded in the past 24 and 12 months, respectively. Median age at most recent viral load was 14 years (IQR: 12–17 years). Approximately equal proportions of adolescents had their most recent viral load recorded at a hospital  and a primary care clinic , with 21.3% (n=187) recorded at a community health centre. At most recent viral load, 74.1% (n=651) were on first-line ART with an NNRTI backbone, and 19.2% (n=169) were on second-line ART with a PI backbone. A further 6.6% (n=58) were on an NRTI-only or unspecified ARV regimen.Among those with clinical records, a further 92.3%  of those with viral load data were virally suppressed at <1000 copies/mL, and 58.4% (n=513) were fully virally suppressed at <50 copies/mL  of the total cohort demonstrated full viral suppression within the past 12 months.At most recent viral load, 76.2% (p=0.026) and sexually infected adolescents  were more likely to have an available clinical record (Table p <0.001) and older age at study enrolment  were associated with lower likelihood of having at least one recorded viral load. Male  and rural-living adolescents  were more likely to have a viral load available in clinical records  and experiencing decentralised care  were associated with having a viral load recorded in the past 12 months (Table p=0.020). However, adolescents who were older at most recent viral load  and rural-residing  were less likely to be virally suppressed at this level. Similarly, adolescents on ART for longer were more likely to be fully virally suppressed at <50 copies/mL , but adolescents who were older at most recent viral load were less likely to be fully virally suppressed . Full results for all model stages for each cascade outcome are provided in Additional Files Longer time on ART , and greater for sexually infected adolescents than their vertically infected counterparts  , and patient-related challenges  , 30. PotHowever, this study has several significant methodological strengths. Notably, participant tracing in the community allowed for correction of facilities’ mortality records. Tracing individual patients across 52 healthcare facilities enabled a more accurate evaluation of adolescents’ health by including unrecorded patient-initiated silent transfers to new facilities , 25. ThiFurther longitudinal analysis of outcomes is required to investigate the cyclical nature of engagement with HIV care, in order to identify when and which adolescents disengage from the HIV care cascade . Future This study documents progression of ART-initiated adolescents across the HIV care cascade to viral suppression in a sub-Saharan African public healthcare setting, accounting for mortality and silent transfers. Adolescents living with HIV in the Eastern Cape, South Africa, demonstrate low rates of viral suppression, and viral load monitoring practices fall well below national guidelines. The effectiveness of UNAIDS 90–90-90 goals for achieving HIV treatment success requires not only viral suppression but also routine, up-to-date viral load monitoring and recording.Additional file 1 Table S1. Full results from sequential multivariable logistic regression analyses of associations between baseline sociodemographic and treatment-related variables and operational HIV care outcomes.Additional file 2 Table S2. Full results from sequential multivariable logistic regression analyses of associations between baseline sociodemographic and treatment-related variables and viral load outcomes.


Many HIV-infected African children gained access to antiretroviral treatment (ART) through expansion of PEPFAR programs since 2004 and introduction of “Test and Treat” WHO guidelines in 2015. As ART access increases and children transition from adolescence to adulthood, treatment failure is inevitable. Viral load (VL) monitoring in Uganda was introduced in 2016 replacing clinical monitoring. However, there’s limited data on the comparative effectiveness of these two strategies among HIV-infected children in resource-limited settings (RLS).> 1 year of first-line ART using only immunologic and clinical criteria to monitor response to treatment were screened in 2010. Eligible children were stratified by VL ≤ 400 and > 400 copies/ml randomized to clinical and immunological (control) versus clinical, immunological and VL monitoring to determine treatment failure with follow-up at 12, 24, 36, and 48 weeks. Plasma VL was analyzed retrospectively for controls. Mixed-effects logistic regression models were used to compare the prevalence of viral suppression between study arms and identify factors associated with viral suppression.HIV-infected Ugandan children aged 1–12 years from HIV-care programs with n = 142) were on NNRTI based ART . One third of ART-experienced children had detectable VL at baseline despite high CD4%. Median age was 6 years (interquartile range [IQR]: 5–9) and 43% were female. Overall, the odds of viral suppression were not different between study arms: , adjusted odds ratio [aOR]: 1.07; 95%CI: 0.53, 2.17, p = 0.57) and did not change over time . Longer duration of a child’s ART exposure was associated with lower odds of viral suppression . Only 13% (9/71) of children with virologic failure were switched to second-line ART, in spite of access to real-time VL.At baseline all children .ClinicalTrials.gov  The 2017 UNAIDS report estimated that 2.1 million children under the age of 15 years were living with HIV, with the majority of these living in Sub-Saharan Africa. Currently in Uganda, an estimated 47% of children living with HIV are receiving antiretroviral therapy (ART) . EffortsMonitoring response to therapy in HIV-infected children has previously been done using CD4 cell counts (immunologic) and clinical criteria because of limited access, lack of technical expertise, inadequate laboratory infrastructure and the high costs associated with viral load testing. This was linked to missed opportunities for timely identification of virologic failure and switch to second-line ART among candidate children (i.e. those who may have benefited from second-line ARV therapy), therefore paving way for emergence of drug resistance –17. In UAlthough there have been some studies done in low income countries that have highlighted the importance of measuring viral load as a more robust method of monitoring response to ART in resource limited settings –28, littWe conducted a two-center, parallel-group randomized clinical trial study in Kampala, Uganda. The study adhered to CONSORT guidelines. HIV-infected children between the ages 1 and 12 years on ART were randomly assigned using a computer-generated list of random numbers (by the study statistician). Randomization was implemented in blocks (size = 4) stratified based on baseline VL (≤ 400 and > 400 HIV RNA copies/ml) to one of two parallel groups - clinical and immunological versus clinical, immunological plus virologic (VL) monitoring for treatment failure with a 1:1 allocation ratio. The allocation sequence was concealed from study staff enrolling participants to prevent selection bias. Study staff involved in the clinical care were aware of a participant’s randomly allocated group due to the nature of study intervention (i.e. monitoring treatment effectiveness) but participants were kept blinded to their group allocation to prevent the likelihood of behavior modification  due to knowledge of group assignment. An independent data safety monitoring committee reviewed unblinded data for patient safety; no interim analyses for efficacy or futility were performed.> Grade 3* (5.1–10 times ULN or greater), and creatinine > Grade 3* (1.9–3.4 times ULN or greater) per DAIDS table for grading the Severity of Adult and Pediatric Adverse Events, Version 1.0, Dated December 2004, Clarification August, 2009.Eligible participants were HIV-infected children aged 1–12 years who (1) had a duration of at least 12 months on a first-line ART therapy and (2) were being monitored under a clinical and immunologic protocol to inform ART regimen change decisions prior to study enrollment. Exclusion was based on the use of second-line ART or PI-based regimen as first-line ART treatment, met immunologic criteria for second-line ART, active TB treatment, liver function tests (AST/ALT) The study took place at two HIV care centers - Makerere University-Johns Hopkins University (MU-JHU) Research Collaboration/MTCT-Plus program and Nsambya Home Care (NHC) in Kampala, Uganda.We enrolled 142 ART-experienced HIV-infected children aged 1–12 years, from an existing treatment program that used clinical and immunologic criteria to monitor response to ART in Kampala, Uganda into the study. The 71 children randomly assigned to each study arm underwent laboratory and clinical evaluations every 12 weeks during the 48 weeks follow-up period. Plasma HIV-1 RNA levels were measured at the MU-JHU Core laboratory with the use of the standard Roche Amplicor v1.5 test kit for the children in the clinical, immunologic & virologic arm.At screening, viral load was assessed but clinicians were blinded prior to randomization. Information on baseline characteristics of the children including; type of ART, prior PMTCT Nevirapine (NVP) exposure, length on ART, clinical and immunological status, and demography were collected. At enrollment and during scheduled follow up visits; medical history, physical examination and information on child’s growth i.e. weight and height, was also collected on standardized study source forms.Adherence monitoring during study follow up was done using self-report by the caretaker and child where applicable, pill counts at the scheduled visits and cross-checked with pharmacy records. For participants who had adherence challenges, unannounced pill counts were conducted during home visits.3 or CD4 percent < 10% for a child aged between 2 and 5 years of age, CD4 count of <100cell/mm3 for a child aged 5 years and above. For Arm 2 this was based on; confirmed viral load > 1000 HIV RNA copies/ml; as well as clinical and immunologic criteria described above. Having 2 confirmed viral loads > 1000 HIV RNA copies/ml at least 2 weeks apart confirmed a virologic endpoint in the study. Clinicians continued participants on first line regimen if viral load is < 5000 HIV RNA copies/ml as per the 2010 WHO guidelines.The children remained on their first-line antiretroviral regimen until they were determined to have treatment failure. Since drug resistance testing was not done in real time, the decision to switch was made based on the assignment of monitoring arm. For Arm 1 this was based on; inadequate weight gain and or increasing WHO clinical staging , CD4 values falling to < 200 cells/mmThe distribution of study participant characteristics in the overall study sample and differences between study arms were examined to validate study randomization procedures respectively.All data analyses were conducted based on the intent-to-treat protocol. Mixed-effects logistic regression models were used to compare the odds  of viral suppression between the two study arms over time. Each child was modeled as a random effect nested within treatment group. Time was modeled as a categorical variable.Changes in the odds  of viral suppression by study arm assignment were examined by testing the significance of the study arm x week interaction term with and without adjustment for potential confounders. In the absence of statistically significant interactions (at a Type I error rate of 0.05), independent study arm, week and other child characteristic effects were examined and reported. A six-step directed acyclic graph (DAG) approach  was usedSimilar procedures described above were used to compare the odds of switching a child to second- line ART regimens between the two study arms and identify correlates of ART change decisions. Missing values in predictor variables were imputed using an R package called MICE 2.22 . MICE fuOverall, 142 children were enrolled in the study and were evenly randomized to either the clinical + immunologic (control) or clinical + immunologic + virologic (intervention) monitoring study arm. The distribution of baseline characteristics did not differ by study arm (Table p = 0.98).Of the 142 participants, 129 (91%) and 134 (94%) completed the 24- and 48-week assessments, respectively. The intervention study arm  had additional assessments at 12 (response rate: 100%) and 36 (response rate: 96%) weeks; however, overall, there were no differences in response rate by study arm (p = 0.63). Overall, the odds of viral suppression were not different between study arms  vs Clinical & Immunologic  and did not change over time [Fig. p = 0.24 and 0 vs 48 weeks: 1.26; 95%CI: 0.92, 1.74, p = 0.15). Having no history of NVP exposure , higher baseline CD4% cell count , 86 (67%) and 93 (69%) children had viral suppression at baseline, 24 and 48 weeks respectively. The prevalence of viral suppression did not differ by study arm during follow-up  virologic testing to standard of care  among treatment-experienced HIV infected children was not associated with a higher prevalence of viral suppression. The results do however support the need for virologic monitoring as a better strategy for early detection of virologic failure especially with increased duration of ART exposure, since immunologic monitoring is a poor predictor of treatment failure. Our findings are in agreement with previous Sub-Saharan Africa studies which have shown that clinical and immunologic monitoring is not sufficient to detect virologic failure in a timely manner , 16, 32,Additionally, our study showed no evidence to suggest virologic testing results in better treatment decisions (i.e. switching to second-line ARVs) linked to better clinical outcomes . Although the odds of switching a child’s ARV regimen were not different between study arms, the odds of switching to second-line regimens increased over time but were not predicted by a child’s treatment exposure history or immunological parameters. Our study highlights the need to operationalize monitoring results into algorithms to inform ARV change decisions since there appears to be a disconnect between monitoring results and actual decisions to switch ARVs.Independent of study arm assignment, having no history of NVP exposure, higher baseline CD4% cell count or absolute CD4 count were associated with higher odds of viral suppression over time while longer duration on ART was associated with lower odds of viral suppression. A similar study done in Tanzania in perinatally HIV infected children  also shoIn contrast to previous studies , 42, ourThe strengths of this study include the longitudinal laboratory and clinical monitoring as well as the high rates of follow up with minimal losses to follow up during the 48-week period. The children in the two arms were comparable in terms of baseline characteristics and virologic suppression at baseline.The limitations of this study include the partial blinding, the short follow up period of 48 weeks and a relatively small sample size of perinatally HIV infected children and adolescents, which may not be generalizable to all ART experienced children. Both the study staff enrolling participants and the participants were blinded to the assignment however, the clinical care staff were not. This could have influenced the behaviour of study staff towards participants. Longer duration of follow up and a larger sample size would confirm the findings of this study. However, the current guidelines and practice recommend that all children on ART should have routine VL monitoring.The results of this study confirm that virologic monitoring is a superior strategy for  detecting early HIV treatment failure especially with the increasing duration of ART exposure among adolescents and lend credence and support to the current WHO guidelines on ART monitoring. The issue of availability and cost of viral load testing still remains a challenge, especially in high burden countries in Sub-Saharan Africa. Therefore, studies to evaluate the affordability and accessibility of these services in resource limited settings will further inform the current WHO recommendations. In addition, there is need to operationalize monitoring results into algorithms to inform ART change decisions since there appears to be a disconnect between monitoring results and actual decisions to switch ART by health workers after ART treatment failure is identified.


Early combination antiretroviral therapy (cART) reduces the size of the viral reservoir in paediatric and adult HIV infection. Very early-treated children may have higher cure/remission potential.in utero (IU)-infected infants in KwaZulu-Natal, South Africa, whose treatment adhered strictly to national guidelines, 76 infants diagnosed via point-of-care (PoC) testing initiated cART at a median of 26 h (IQR 18–38) and 75 infants diagnosed via standard-of-care (SoC) laboratory-based testing initiated cART at 10 days (IQR 8–13). We analysed mortality, time to suppression of viraemia, and maintenance of aviraemia over the first 2 years of life.In an observational study of 151 p<0.0001) and with concurrent maternal cART failure . High-frequency virological failure was observed in PoC- and SoC-tested groups of children.Baseline plasma viral loads were low (median 8000 copies per mL), with 12% of infants having undetectable viraemia pre-cART initiation. However, barely one-third (37%) of children achieved suppression of viraemia by 6 months that was maintained to >12 months. 24% had died or were lost to follow up by 6 months. Infant mortality was 9.3%. The high-frequency virological failure in IU-infected infants was associated not with transmitted or acquired drug-resistant mutations but with cART non-adherence (plasma cART undetectable/subtherapeutic, The success of early infant testing and cART initiation strategies is severely limited by subsequent cART non-adherence in HIV-infected children. Although there are practical challenges to administering paediatric cART formulations, these are overcome by mothers who themselves are cART-adherent. These findings point to the ongoing obligation to address the unmet needs of the mothers. Eliminating the particular barriers preventing adequate treatment for these vulnerable women and infants need to be prioritised in order to achieve durable suppression of viraemia on cART, let alone HIV cure/remission, in HIV-infected children.Wellcome Trust, National Institutes of Health. We searched PubMed up to May 31, 2019 for original and review articles, on HIV-infected infants; the timing and outcome of treatment, by searching the terms “HIV”, “antiretroviral therapy”, “infant treatment” and “outcome” or “suppression” or “early”, not limited by language. It has been well established that for perinatally HIV-infected infants, early cART initiation reduces morbidity, mortality, and viral reservoir size, suggesting the potential for cure/remission in this group. A recent study showed that cART initiation within the first 48 h of life for IU-infected infants is feasible in South Africa. However, for reasons unknown, it did not improve mortality, retention in care, or viral suppression rates in infancy.We studied a large, contemporary cohort of IU-infected infants in KwaZulu-Natal, South Africa. Pre-cART plasma HIV RNA and DNA viral loads were low. However, cART failure rates were high. In the subset of children tested, this was not due to transmitted or acquired cART resistance. High-frequency cART failure was due to cART non-adherence in the children.Where antenatal cART is available, IU-MTCT occurs in a vulnerable subset of mothers for whom cART self-administration, and administration to their infected child, is difficult to achieve. In order to maximise health in perinatally infected children, as well as the high potential for HIV cure/remission, early infant diagnosis and cART initiation alone is insufficient without meeting the socioeconomic needs of transmitting mothers. There is an urgent need to address the particular challenges facing mothers of HIV-infected infants, and at the same time to explore long-acting therapeutic options to lessen the medication burden for effective anti-HIV treatments for children.Alt-text: Unlabelled box1Initiation of combination antiretroviral therapy (cART) early in HIV infection reduces the size of the viral reservoir, estimated by the frequency of HIV DNA in peripheral blood cells, both in paediatric and adult infection in utero (IU) infection There is some albeit limited evidence that the potential to achieve cure/remission may be greatest in children following Several anecdotal cases of cure/remission in early cART-treated children further support the notion that initiation of cART within the first days of life might pave the way to achieving cure/remission in a substantial proportion of those treated 22.1Ucwaningo Lwabantwana (meaning ‘Learning from Children’) is an ongoing observational prospective cohort study designed to determine the feasibility of very early (<48 h of life) cART initiation for IU HIV-infected infants. In 2015, the study began to identify IU HIV-infected infants from four secondary-level hospitals in KwaZulu-Natal, South Africa. In this setting, there is lifelong cART for pregnant women and routine infant birth HIV total nucleic acid (TNA) PCR.Baseline results from this current cohort were compared to IU HIV-infected infants from a historical cohort set in Durban in KwaZulu-Natal in 2002–2005  ,15. At t2.2These studies were approved by the Biomedical Research Ethics Committee of the University of KwaZulu-Natal and the Oxfordshire Research Ethics Committee. Written informed consent for the infant and mother's participation in the study was obtained from the mother or infant's legal guardian.2.3High-risk mother: one or more of the following criteria during pregnancy; maternal seroconversion, <4 weeks of maternal cART prior to delivery, suboptimal cART adherence by history or a documented plasma viral load >1000 HIV RNA copies per mL.Maternal seroconversion: a documented negative rapid HIV-1 antibody test in pregnancy followed by a positive result later in pregnancy or at delivery.cART non-adherence: missing three or more consecutive cART doses.In utero HIV-infected: an infant with a positive or indeterminate TNA PCR taken <48 h of age that was later confirmed positive via further testing from a separate blood samples i.e. in all cases diagnosis required two or more positive nucleic acid tests.Viral suppression: Plasma HIV RNA level below the limit of detection (<20 or <100 HIV RNA copies per mL depending on sample volume) on one occasion.Viral rebound: Plasma HIV RNA >1000 copies per mL on one occasion or two consecutive measurements >100 HIV RNA copies per mL following viral suppression.Loss to follow-up: an infant that did not return to the study site for ≥6 months despite active tracing.2.4For the Ucwaningo Lwabantwana study, HIV-positive mothers were screened after delivery to assess risk of IU HIV transmission. Infants of high-risk mothers were tested for HIV-1 as soon as possible after birth using PoC TNA PCR on whole blood  or where PoC testing was not available, by overnight plasma HIV RNA viral load measurement . The results of these tests were binary with no indeterminate range.Infants from the same hospitals who either declined PoC testing or who were discharged before they could be tested, and those born at the hospitals’ local clinics with a positive or indeterminate result from the standard-of-care (SoC) laboratory-based dried blood spot TNA PCR  at birth were also enroled. Turnaround time for the SoC test result was approximately 4–7 days. In all cases diagnosis required two or more positive HIV nucleic acid tests. Eligibility criteria for enrolment via any method of testing included an IU HIV-infected infant initiated on cART ≤21 days of life.All HIV exposed infants were given prophylactic ART as soon as possible after birth, consisting of at least daily NVP, and additional twice daily zidovudine (AZT) for infants deemed high risk of HIV-1 infection, in accordance with the South African National PMTCT Guidelines. After the first positive infant HIV-1 test result was received, blood for confirmatory testing and baseline measurements was taken and cART consisting of twice daily NVP, AZT and lamivudine (3TC) was initiated as per local guidelines. This neonatal regimen was switched to ritonavir-boosted lopinavir (LPVr), 3TC and abacavir (ABC) at 42 weeks corrected gestational age or at 1 month of age.T cell quantification, plasma viral load , and storage of peripheral blood mononuclear cell (PBMC) and plasma.Mother and infant follow-up occurred monthly for 6 months then 3 monthly. At each visit, blood was drawn for CD4+ During follow-up, additional support was provided for the caregivers, including regular one-on-one counselling sessions from trained HIV counsellors in the local language (isiZulu), paid transport to clinic, appointment reminders via phone, family support groups and home visits where required.Stored PBMC and plasma samples were used for additional confirmatory testing, and where available, for the quantification of drug resistance mutations (DRMs) by Next Generation sequencing methods, HIV DNA by droplet digital PCR , and plasma ART levels by high performance liquid chromatography coupled to tandem mass spectrometry (Supp. Methods).2.5To assess the feasibility and impact of very early cART initiation in this current cohort, Ucwaningo Lwabantwana, we considered outcomes for the whole cohort of IU HIV-infected infants together and then compared two subgroups of IU-infected infants according to the timing of test they were first identified by: the PoC and SoC groups. Comparisons included: time to cART initiation, maternal and infant baseline characteristics, time to viral suppression, viral rebound rates, mortality, loss to follow-up and viral suppression rates at 12 and 24 months. To assess the influence of drug resistance on virological outcomes, DRMs were quantified and plasma ART levels were measured to evaluate potential non-adherence to treatment. To assess the impact of the current PMTCT programme, these infants’ baseline results were also compared to those infants from the historical PEHSS cohort.2.6ξ was determined via 10-fold cross validation using the glmnet R package Clinical and laboratory results were described using absolute numbers, percentages, medians and interquartile range (IQR). Comparisons were performed using the Chi-Squared and Fisher's exact tests for categorical variables and the Mann-Whitney U test for continuous variables. Mortality rate, viral suppression and viral rebound were calculated in univariate analyses using Kaplan-Meier curves and groups were compared using the Log-Rank test. Because of the large number (25) of candidate mother-child variables at baseline, predictors of mortality, viral suppression and viral rebound were analysed in multivariate analyses using a penalised Cox regression model, where the set of relevant predictors was selected by the LASSO penalty approach 3The funders of this study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.4n = 70) or overnight viral load (n = 6) testing of infants, and cART was initiated at a median age of 26 h (IQR 18–38). A further 75 infants were diagnosed through SoC testing, in whom cART was initiated at a median age of 10 days (IQR 8–13). ART prophylaxis did not differ significantly between the PoC and SoC groups  of the IU-infected infants were born to mothers designated as seroconverting during the pregnancy. This included 41 mothers who had a negative HIV test during pregnancy followed subsequently by a positive test; and 6, in whom the estimated seroconversion date, midway between last negative test and first positive test, was also during pregnancy. In 28% (42/151) of cases, the infants were born to mothers known to have been HIV-infected prior to conception, including 6 who were themselves infected via MTCT . The remp = 0.011)  . However= 0.011) .T cell count in the PoC and SoC groups were taken, respectively, at a median of 1 day and 10 days of age, and thus after 1 and 10 days of prophylactic ART . As expected, therefore, at baseline, the SoC infant group had significantly lower plasma viral loads than the PoC group , higher CD4+ T cell counts  and higher CD4+ T cell percentage,  measurements of plasma viral load and CD4+ appeared A–C.Fig. T cell counts significantly higher in the current Ucwaningo Lwabantwana cohort of 151 IU-infected infants compared to the 2002–2005 PEHSS cohort of 50 IU-infected infants, also in KwaZulu-Natal, South Africa . It is noteworthy that as many as 19/151 (12.5%) of the Ucwaningo Lwabantwana cohort of IU-infected infants had undetectable plasma viral loads (<20 HIV RNA copies per mL or <100 HIV RNA copies per mL) at baseline, consistent with the notion that, in the majority of IU-infected infants, there is significant intrauterine ART exposure transplacentally.Baseline infant plasma HIV viral loads were significantly lower and absolute CD4+ h Africa D–F, when10 higher in the PoC group, HIV DNA viral loads measured at the same timepoint showed minimal difference between the groups , or were lost to follow-up  (n = 151), 15 infants died. The probability of death by age 3 months was 6.4% (95% CI 2.3–10.3%) and was 9.3% by 1 year  with a median age of death of 3.5 months. The multivariate analysis identified three independent predictors of mortality: low infant birthweight; neonatal hospital admission and high infant baseline plasma HIV RNA viral load (Supp. Tab. 1).To assess initial outcome in SoC and PoC-treated, IU-infected infants, we next evaluated the 114/151 infants from the Ucwaningo Lwabantwana cohort who were born ≥12 months prior to the time of analysis. Excluding four infants (two PoC- and two SoC-tested) who transferred out in the first week of life to receive care at external clinics, 26 of the remaining 110 (24%) had either died  A. amongsOf the infants retained on the study at 12 months of age, 15/110 (13.6%) had not achieved suppression of plasma viraemia on cART by 6 months and 28/110 (25.5%) had achieved only short-term suppression of viraemia, rebounding by 12 months A. Only 3p = 0.75, and p = 0.19, respectively), in the univariate analyses, infants in the SoC group tended to achieve suppression of viraemia earlier, and to maintain suppression of viraemia longer than those in the PoC group . These findings held true in the multivariate analyses, with a strong predictor at baseline for rebound being the PoC group compared with the SoC group, Supp In order to further investigate why sustained suppression of viraemia in early-treated infants on ART is so infrequent, we first compared the PoC and SoC groups. Although there was no difference in mortality or retention on the study at 12 months  were deep sequenced for DRMs at baseline  were transmitted from the mother to the child. Samples from a subset of infants  aged >6 months  with virological failure, defined as a plasma RNA viral load of >1000 HIV RNA copies per mL. We also measured levels of antiretroviral drugs in these 16 plasma samples, since although ART detection reflects adherence only in the previous 24–48 h, ART non-detection unequivocally demonstrates non-adherence during this window . All 11 of the virally suppressed infants had all three prescribed ART drugs detectable at high levels in the plasma, while 13/16 (81%) of the infants with high viral loads had either no or only one ART drug detected at therapeutic levels  Finally, in 101 children older than 6 months of age who had a plasma viral load measurement at the same time point as their mother, viral suppression in the mother and child was strongly associated  of cART-treated infants achieved suppression of viraemia by 6 months that was maintained beyond 12 months of age. Almost one quarter (24%) of IU-infected infants either died or were lost to follow-up by 6 months. In the subset tested, high-frequency cART failure did not result from drug-resistant mutations (DRMs). The barriers contributing to treatment failure are the same in both mother and child. This is an important result because it means that, if the needs of the mother can be successfully addressed, the practical challenges of administering cART to children, in most cases, will also be met. If durable suppression of viraemia in HIV-infected infants is to be achieved, and the undoubted opportunities to achieve cure/remission in HIV-infected children are to be fully realised, the unmet needs of transmitting mothers urgently need to be addressed first, whilst, at the same time, exploring options for long-acting or extended-release paediatric ART formulations. Finally, apparently superior treatment outcomes for the SoC tested group suggests that barriers to returning to clinic to collect test results selects out infants with inherently better outcomes, and that although PoC testing gives the opportunity to capture these vulnerable infants, more focus needs to put into supporting the maintenance of cART once initiated.In South Africa, IU-infected infants usually receive cART transplacentally in addition to ART prophylaxis  immediately after birth (prior to HIV diagnosis). In this setting, neither this study nor another South African study by Technau et al. in utero, being more likely to have difficulty administering infant cART compared to mothers who transmitted simply because they were diagnosed late in pregnancy. Interestingly, infants born at a younger gestational age and also those admitted to hospital as a neonate had lower rates of rebound, suggesting that additional support for the mother in the first weeks of life may be protective for virological control over the longer term. Older maternal age was also found to be protective against infant viral rebound. The importance of considering a child in the context of the mother is nowhere better illustrated than in the setting of perinatal HIV.The high rates of treatment failure we present here, along with other studies describing the difficulties in treating paediatric HIV infection in sub-Saharan Africa , as wellInfant mortality at 1 year was 9.3%, similar to other studies of South African IU HIV-infected infants It is important to consider the potential limitations within this study. Most crucially, in our comparisons of outcomes following PoC testing versus SoC testing, it should be emphasised that this was not a randomised controlled trial. Indeed, there were some differences between the PoC and SoC groups that were unexpected, such as the proportion of females (74% vs 53%), and the proportion exclusively breast-fed (91% versus 68%), the clinical significance of which were unclear. With these differences at baseline, and the outcomes of the SoC tested infants who were not recruited onto the study unknown, a direct comparison of the PoC and SoC infants should be interpreted with caution. However, what is irrefutable, is that despite very early cART initiation, PoC tested infants’ outcomes are poor. It has been suggested that integrase strand inhibitor drugs can achieve suppression in early treated infants faster ART has been widely successful in improving health, prolonging life and reducing HIV transmission events across sub-Saharan Africa. Furthermore, cART adherence in acutely infected women from a similar sociodemographic in KwaZulu-Natal with additional support can achieve excellent outcomes JRM coordinated the clinical aspects of the study, analysed the data and wrote the manuscript. NB, RF, KS, VN, NK, JR, KD, PK undertook the clinical management of the subjects, provided the clinical samples and contributed to writing the manuscript. VAV, MA, MM, AG, NG, PCM, TN, JA, KG, DB, TC, MB, NN, NI, EA, BDW, MCP and JMP undertook data analysis and helped in writing the manuscript. PG conceptualised and led the study, analysed the data and assisted in writing the manuscript. From the Ucwaningo Lwabantwana Consortium JvL, YG, KC, CK, RNB, MK and NM assisted with the identification of study subjects, provision of clinical samples for the study and writing of the manuscript, and RB, GM, and MdC undertook data analysis and helped in writing the manuscript.Dr. Martinez-Picado reports institutional grants and educational/consultancy fees outside the submitted work from AbiVax, Astra-Zeneca, Gilead Sciences, Grifols, Janssen, Merck and ViiV Healthcare. Dr. Millar reports personal fees from Cepheid, outside the submitted work.