Antiretroviral therapy (ART) scale-up in sub-Saharan Africa combined with weak routine virological monitoring has driven increasing HIV drug resistance. We investigated ART failure, drug resistance, and early mortality among patients with HIV admitted to hospital in Malawi.This observational cohort study was nested within the rapid urine-based screening for tuberculosis to reduce AIDS-related mortality in hospitalised patients in Africa (STAMP) trial, which recruited unselected  adult (aged ≥18 years) patients with HIV-1 at admission to medical wards. Patients were included in our observational cohort study if they were enrolled at the Malawi site  and were taking ART for at least 6 months at admission. Patients who met inclusion criteria had frozen plasma samples tested for HIV-1 viral load. Those with HIV-1 RNA of at least 1000 copies per mL had drug resistance testing by ultra-deep sequencing, with drug resistance defined as intermediate or high-level resistance using the Stanford HIVDR program. Mortality risk was calculated 56 days from enrolment. Patients were censored at death, at 56 days, or at last contact if lost to follow-up. The modelling strategy addressed the causal association between HIV multidrug resistance and mortality, excluding factors on the causal pathway .Of 1316 patients with HIV enrolled in the STAMP trial at the Malawi site between Oct 26, 2015, and Sept 19, 2017, 786 had taken ART for at least 6 months. 252 (32%) of 786 patients had virological failure . Mean age was 41·5 years (SD 11·4) and 528 (67%) of 786 were women. Of 237 patients with HIV drug resistance results available, 195 (82%) had resistance to lamivudine, 128 (54%) to tenofovir, and 219 (92%) to efavirenz. Resistance to at least two drugs was common , and this was associated with increased mortality .Interventions are urgently needed and should target ART clinic, hospital, and post-hospital care, including differentiated care focusing on patients with advanced HIV, rapid viral load testing, and routine access to drug resistance testing. Prompt diagnosis and switching to alternative ART could reduce early mortality among inpatients with HIV.Joint Global Health Trials Scheme of the Medical Research Council, UK Department for International Development, and Wellcome Trust. Despite the unprecedented scale-up of antiretroviral therapy (ART) in high-HIV prevalence settings in sub-Saharan Africa, HIV remains a common cause of admission to hospital, with high early mortality (31% in the African region).6HIV drug resistance is increasingly common in sub-Saharan Africa, with recent estimates of 10–15% prevalence for resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) in untreated patients (transmitted drug resistance), and much higher prevalence (50–80% for NNRTIs) in patients who have had ART.Evidence before this studyWe searched MEDLINE for studies that investigated virological failure or HIV drug resistance in patients with HIV on antiretroviral therapy (ART) who are hospital inpatients in Africa, published between Jan 1, 2005, (ART was not in widespread use before this date), to Jan 1, 2020. We combined search terms for HIV  with terms for virological failure  or resistance , and with terms for hospital inpatient  and Africa. We only identified one observational cohort study that reported prevalence of virological failure in two cohorts of inpatients. This study showed a high prevalence of patients who were already taking ART but had HIV-1 viral load of at least 1000 copies per mL. However, the study did not do viral load testing on all patients taking ART, introducing bias. There were no studies reporting HIV drug resistance among hospital inpatients. Other studies identified all reported data from outpatient clinics.Added value of this studyWe did HIV viral load testing on unselected patients admitted to hospital who were taking ART for at least 6 months, and looked for HIV drug resistance mutations for patients who had high HIV viral loads. We then assessed whether the presence of drug resistance was associated with mortality. Approximately one-third of inpatients taking ART had virological failure, and most of these patients had drug resistance to first-line ART which was associated with increased early mortality. These are the first data on HIV drug resistance in hospital inpatients from high-burden settings in Africa that show the high prevalence of HIV drug resistance.Implications of all the evidenceART failure and drug resistance is a major problem for hospital inpatients in settings with high HIV prevalence. Our results support testing hospital inpatients for ART failure, ideally using rapid HIV-1 viral load assays so that results are available quickly and can be immediately acted upon. The high prevalence of drug resistance and association with early mortality suggests these patients should be switched to alterative ART, and supports development of low-cost, rapid assays to detect HIV drug resistance. Differentiated ART clinic care to support adherence and detect ART failure in advanced HIV, testing and screening for opportunistic infections, and improved care post discharge could improve outcomes, although further evidence for such interventions will be needed.Although most available data on HIV drug resistance come from outpatient clinics, inpatients repwresent a key target group for intensified interventions, given their relatively high risks of treatment failure, advanced immunosuppression, and high short-term mortality.To describe the prevalence of virological failure and HIV drug resistance, and their effect on early mortality in patients with HIV admitted to hospital in high-HIV burden settings, we carried out an observational cohort study nested within a large tuberculosis screening trial in sub-Saharan Africa.This cohort study was nested within the rapid urine-based screening for tuberculosis to reduce AIDS-related mortality in hospitalised patients in Africa (STAMP) trial, which recruited unselected , adult (aged ≥18 years) patients with HIV-1 at admission to medical wards.Exclusion criteria were recent tuberculosis treatment (past 12 months), recent tuberculosis preventative therapy (past 6 months), or being unable or unwilling to provide informed consent. The study team recorded clinical data at admission and during hospitalisation on standardised case report forms based on patient interview, medical records, and clinical review. Patients discharged alive were followed up at 56 days. Vital status was established by home visit, telephone, or through next of kin for those not attending follow-up.Patients were included in this study if they were enrolled at the Malawi site  viral load assay  in Malawi. For enrolled patients with virological failure  HIV-1 genotyping to detect drug resistance mutations was done by ultra-deep sequencing in the UK.Iterative Virus Assembler. Following sequencing, samples were aligned using the MAFFT program (version 7).Nucleic acids were extracted from 230 μL of plasma using the DSP Virus/Pathogen kit on the QIAsymphony platform  and amplified using in-house HIV primer sets . Library preparations were generated using the Nextera XT DNA Sample Preparation Kit and sequenced on the Illumina MiSeq platform . Bioinformatic analysis was done using the de novo Stanford HIVDB program (version 2.3.0).HIV-1 subtype and drug resistance mutations were analysed using the 2 tests, continuous data using t tests or Wilcoxon rank sum dependent on distribution. Mortality risk was calculated 56 days from enrolment. Patients were censored at death, at 56 days, or at last contact if lost to follow-up. Cox proportional hazards models were used to assess associations with mortality, p values were calculated using likelihood ratio tests. The modelling strategy addressed the causal association between HIV multidrug resistance and mortality, excluding factors on the causal pathway . All models were adjusted for STAMP trial arm.Categorical data were compared using χIn the Cox regression analysis, linear association and departures from linearity of continuous variables with log  were assessed using the likelihood ratio test. For the mortality regression analysis, all patients with viral load below 1000 copies per mL were assumed to have no drug resistance, and patients with virological failure but missing drug resistance data were excluded. A sensitivity analysis was done only including patients on ART for more than 12 months. Results are reported as hazard ratios (HRs) with 95% CIs and Kaplan-Meier curves. We used Stata  for analyses. See The funder of the 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.Of 1316 patients with HIV enrolled in the STAMP trial at the Malawi site between Oct 26, 2015, and Sept 19, 2017, 1108 (84%) knew their HIV status. Of those who knew their HIV status, 1021 (92%) were taking ART, with 814 (80%) on ART for at least 6 months at hospital admission. 28 patients had missing HIV viral load measurements, leaving 786 included in the analysis . Mean ag468 (60%) of 786 patients had undetectable HIV-1 viral loads (<50 copies per mL) and 252 (32%) had virological failure  with a median viral load of 125 603 copies per mL. A further 66 (8%) had low-level viraemia (50–999 copies per mL). Patients with virological failure were younger, more likely than other patients to be male, and more likely to have lower body-mass index (BMI), Karnofsky scores, and CD4 cell counts , with 236 (94%) having advanced HIV at admission. They were also more likely to receive antimicrobial and tuberculosis treatment as inpatients, and had a longer median length of hospitalisation  were successfully sequenced  and all were HIV subtype C. 221 (93%) of 237 patients had drug resistance mutations to first-line or second-line drugs.The most common relevant nucleoside reverse transcriptase inhibitor (NRTI) drug resistance mutations were Met184Val  and Lys65Arg/Asn  samples resistant to efavirenz, and 220 (93%) resistant to nevirapine. The most common NNRTI mutations were Lys103Asn/Ser/His (40%), Tyr181Cys/Ile/Glu (37%), and Gly190Ala/Ser/Glu (41%). Resistance to newer NNRTI drugs was also common and consistent with anticipated cross-resistance, with 158 (67%) samples resistant to rilpivirine, and 146 (62%) resistant to etravirine.Protease inhibitor and integrase inhibitor resistance were rare, with only one (<1%) of 233 patients having a major protease inhibitor mutation , and two (1%) patients having accessory protease inhibitor mutations (Gln58Glu). Two (1%) of 225 patients also had major integrase inhibitor mutations (one had Gln148Gln/His and one had Thr66Ser), and eight (4%) had accessory integrase inhibitor mutations (six had Glu157Gln and two had Gln95Lys).Overall, only 18 (8%) of 237 patients had no detectable resistance to any first-line ART drugs, with 127 (54%) having resistance to all first-line drugs, and 196 (83%) to at least two drugs . Assuminvs 9% [44 of 534], 95% CI 7–12% in those without; p=0·0020), with inpatient mortality similar for those with virological failure (23 [9%] of 252) and those without (50 [9%] of 534).Mortality by day 56 was 20% , with 83 (53%) of 156 deaths occurring after discharge from hospital . MortaliAmong 237 patients with virological failure and available HIV drug resistance genotypes, unadjusted mortality increased with increasing drug resistance. Mortality by day 56 was 6% in patients with no drug resistance, 13% in patients with resistance to one drug, and 28% in patients with multidrug resistance p=0.041; .In analyses adjusted by STAMP trial arm only, age, sex, time on ART, advanced HIV, BMI, Karnofsky score, CD4 count, haemoglobin, WHO danger signs, tuberculosis treatment, and virological failure were all strongly associated with increased mortality . HIV mulIn exploratory analyses, adjusting for CD4 cell count, clinical signs of advanced HIV, and poor functional or nutritional status mitigated the association of HIV multidrug resistance and mortality, supporting their position on the causal pathway between multidrug resultant HIV and death, and their exclusion from the multivariable causal model.79 samples from patients not taking ART at hospital admission were successfully sequenced. 60 of those patients were ART naive, and 19 had previously taken ART. Baseline characteristics differed , with paOur findings show that, in Malawi, most inpatients with HIV knew their HIV status and were taking ART, in contrast with previously reported hospital cohorts.9To our knowledge, this is the first study to report virological failure and HIV drug resistance in unselected patients with HIV admitted to hospital in sub-Saharan Africa, and to report mortality outcomes. Hospital inpatients are an important source of information on the major causes of severe illness and death in key subpopulations such as people living with HIV. Consistent with regional HIV care cascade data, our data show undiagnosed and untreated HIV was a less common cause of severe illness than previously reported, given the high proportion of patients already taking ART at admission.Our finding that 19% of all admissions among people living with HIV (25% of patients taking ART for any duration) had virological failure highlights the growing importance of drug resistance and system weaknesses that limit diagnosis and management of virological failure before the onset of critical illness. Other African data also show widespread ART failure among inpatients, and to a lesser extent among outpatients.In our study, virological failure was synonymous with HIV drug resistance. NNRTI resistance occurred in 92% of patients, an unsurprising observation given widespread use and low barrier to resistance, but resistance was more common than in outpatient studies.Mortality in this cohort was high: 20% patients died by day 56. We found markers of advanced HIV disease were associated with mortality. HIV drug resistance was also associated with mortality, showing a dose-response relationship, and multidrug resistance was independently associated with mortality. Viral load testing is rarely done for inpatients both generally and in Malawi specifically, and centralised testing programmes tend to have long turnaround times and lack electronic laboratory management systems, causing patients to either have died or been discharged before results are available.Our findings suggest interventions aimed at preventing and diagnosing virological failure and drug resistance could reduce morbidity and mortality in patients taking ART. Before hospital admission, patients were actively attending ART clinics that did not identify and address their ART failure and advanced HIV. Although the public health approach to ART has led to declines in HIV incidence and mortality, patients with advanced disease (despite engagement with clinics) would benefit from differentiated care, with a focused approach to patients with high viral loads and more frequent viral load testing, review, and monitoring.Currently, HIV drug resistance testing has not been prioritised by HIV programmes in high-burden settings in sub-Saharan Africa. Point-of-care technologies to detect important drug resistance mutations are in the pipeline, and their use to guide ART choice has led to improved outcomes among patients with pre-treatment drug resistance before initiation of first-line ART.In hospital, patients taking ART who have signs of advanced HIV would benefit from rapid, near-patient testing for virological failure, for example using the Xpert HIV-1 viral load assay . Its use was recently shown to increase viral suppression and retention in community-based care in South Africa.Malawi and WHO guidelines for ART introduced dolutegravir-based regimens (with tenofovir and lamivudine NRTI backbone) as the preferred first-line ART from 2019.The strengths of our study are that it is a large cohort nested in a clinical trial, and unselected patients with HIV were enrolled, reducing bias. Furthermore, the data reflect routine clinical care in hospitalised patients. There are also some limitations. It is a single-centre study, although results are likely to be generalisable to other settings in sub-Saharan Africa. There is also missing data on HIV viral loads and HIV drug mutations. The exclusion of patients not consenting might have underestimated virological failure and multidrug resistance, as these people are likely to be more unwell. We assumed that patients with viral loads below 1000 copies per mL had no drug resistance, but the prevalence and implications of resistance in this group is not clear. We did not have detailed adherence information for patients. However, patients all self-reported taking ART and had evidence of attending ART clinics and ART being dispensed. Furthermore, high prevalence of drug resistance also supports that patients were taking drugs, as reversion to wild type is common without drug pressure.In conclusion, we have found virological failure and HIV drug resistance to be extremely common in inpatients with HIV, and drug resistance was associated with increased mortality. Patients already established on ART with advanced HIV disease need screening for failure during hospital admission, ideally using rapid assays. Those identified as having ART failure would likely benefit from switching to alternative ART . Interventions targeting ART clinic and post-hospital care are also needed. Our findings support the development of low-cost and rapid assays to detect HIV drug resistance.


Migration is associated with HIV-1 vulnerability. Objectives: To identify long-term trends in HIV-1 molecular epidemiology and antiretroviral drug resistance (ARV) among migrants followed up in Portugal Methods: 5177 patients were included between 2001 and 2017. Rega, Scuel, Comet, and jPHMM algorithms were used for subtyping. Transmitted drug resistance (TDR) and Acquired drug resistance (ADR) were defined as the presence of surveillance drug resistance mutations (SDRMs) and as mutations of the IAS-USA 2015 algorithm, respectively. Statistical analyses were performed. Results: HIV-1 subtypes infecting migrants were consistent with the ones prevailing in their countries of origin. Over time, overall TDR significantly increased and specifically for Non-nucleoside reverse transcriptase inhibitor (NNRTIs) and Nucleoside reverse transcriptase inhibitor (NRTIs). TDR was higher in patients from Mozambique. Country of origin Mozambique and subtype B were independently associated with TDR. Overall, ADR significantly decreased over time and specifically for NRTIs and Protease Inhibitors (PIs). Age, subtype B, and viral load were independently associated with ADR. Conclusions: HIV-1 molecular epidemiology in migrants suggests high levels of connectivity with their country of origin. The increasing levels of TDR in migrants could indicate an increase also in their countries of origin, where more efficient surveillance should occur. In the latest years, the number of migrants has dramatically increased globally . Almost In Portugal, migrants account for 32% of all new HIV diagnoses . NotablyThe global distribution of subtypes and circulating recombinant forms reflects the complexity of the molecular epidemiology of HIV-1, which might be affected by human mobility, including the different patterns of mobility: local migration, short journey migrants, migration by stages, long-journey migrants and temporary migrants . MigrantThe introduction of highly active antiretroviral therapy (HAART) into developed countries in 1996 has radically changed the clinical outcome of HIV, leading to decreased mortality and morbidity with HIV-1 infection ,8,9. HowThe prevalence of people living with HIV in Portugal has been increasing continuously, and concomitantly the proportion of patients treated with antiretrovirals has also been increasing ,13.In the 16th century Portugal colonized several countries, including countries in Africa , as well as Brazil in South America. HIV molecular epidemiology studies are crucial in these areas to prevent the widespread transmission of drug resistance, especially those lacking systematic surveillance as Portuguese Speaking African Countries (PALOP). The main objective of this study was to analyze the prevalence and patterns of transmitted antiretroviral drug resistance (TDR) and acquired drug resistance (ADR) and the current circulating HIV-1 variants among HIV-1 infected patients migrants originating from Portuguese speaking countries, followed-up in Portuguese hospitals. To achieve this, we combined socio-demographic data, clinical and of viral sequences sampled from migrants followed in Portuguese hospitals and compared it with a dataset of Portuguese sequences available at the same time period of the study.The study population included 5177 HIV-1 positive patients independent of their AIDS status. Eligible participants were adults older than 18yo who attended the HIV/AIDS service from Portugal. Patients were included if they had at least one available genotypic resistance profile between January 2001 and December 2017. Overall, 1490 were migrants, and 3687 were Portuguese individuals followed up during the same time aforementioned. Sociodemographic , clinical (CD4+ T cell count) and virological  data were linked to partial pol gene sequences  and reverse transcriptase, codons 1-247). Data was extracted from the central RegaDB resistance database hosted at the Egas Moniz hospital  [pol gene (PR/RT) were performed by different laboratories in whole country using in-house and/or commercial drug resistance tests. HIV-1 subtypes were determined by REGA HIV-1 Subtyping Tool [http://jphmm.gobics.de/submissionhiv.html) [https://comet.lih.lu/) [Part of ing Tool ,16 softwiv.html)  and Contlih.lu/) .https://hivdb.stanford.edu). In order to access Acquired Drug Resistance (ADR), the Genotypic Resistance Interpretation Algorithm of the HIVdb program (http://sierra2.stanford.edu/sierra/servlet/JSierra) was used. The HIVdb program was also used to infer the resistance profile of the HIV-1 sequences and its clinical impact score. The Stanford algorithm comprises mutations contained in the IAS-USA drug resistance mutation list and classifies isolates as susceptible/potential (S), low (L) intermediately (I) or high (H). It was estimated according to the HIVdb Interpretation Algorithm version 8.4 .Pol sequences generated by sanger sequencing population were analyzed on Stanford CRP V.6.0 tool to detect for surveillance drug resistance mutations (SDRMs), according to the WHO 2009 SDRM list . The prep < 0.05 were retained for adjusted analyses. The variables included in the adjusted analysis were age, country of origin, subtype, VL and sampling year. All statistical associations were considered significant if p < = 0.05. Statistical analyses were conducted using SPSS and on R.Descriptive statistics for continuous variables of HIV-1 infected individuals subjects were calculated as frequency (percentage) and median Interquartile ranges (IQR:25%-75%). Differences between group were calculated by Mann–Whitney U test (MWT) and the Kruskal-Wallis. Proportions were given with a 95% confidence interval (CI) based on binomial distribution. Differences in proportions were assessed by chi-squared test. we divided patients into 4 groups by date of sampling (2001–2008 vs. 2009–2011 vs. 2012-2014 vs. 2015–2017). Simple logistic regression of global TDR and each class of drugs was performed. Simple and multiple binary logistic regression models were also performed to identify possible factors associated with TDR and ADR. The variables included: age, country, subtype, gender, CD4+, VL and sampling year. Variables with All analyses were performed anonymously. This study was approved by the ethical committee of Egas Moniz hospital . All procedures performed in studies involving human participants were in accordance with the ethical standards of the Clinical Research ethical committee of Egas Moniz Hospital (108/CES-2014 – 15-10-2014) and with the Helsinki declaration. It was designed to protect the rights of all subjects involved under the appropriate local regulations.p = 0.044), median of CD4+ T cell counts was 323 (133–498) cells/μL and 244 (121 – 408) cells/μL (p < 0.001) and finally HIV viral RNA load was 4.8 (4.1–5.3)log copies/mL and 4.33 (3.6–4.9)log copies/m (p < 0.001) on the other hand, there were no significant differences between gender and age. The characteristics of the participants included in the analysis are summarized in A total of 5177 HIV-1 sequences were included in the analysis and consisted of 1281 (24%) of HIV-1 adult migrants from Portuguese-speaking African countries (PALOP), 209 (4%) from Brazil and 3687 (72%) Portuguese-originated patients, followed up between 2001 and 2017. Overall, 3552 (69%) naïve patients and 1589 (31%) were adhering to a therapeutic regime had complete RT and PR sequences. The number of patients per year varied from 55 to 523 since 2001–2017, including 1839 (35.5%) women and 3294 (63.4%) men with a median age of 39 years (range 32–49). More than 60% of patients had viral load measured and the median plasma HIV RNA was 4.64 log10 copies/mL (3.9–5.2), and the median CD4 count was 281cells/μL (range 128–461 cells/μL). Significant differences between subjects without vs. with previous treatment were found in geographical origin of samples (p < 0.001). The epidemic among PALOP is driven by female (53%). However, in the Brazilian migrants and Portuguese groups, females are the minority: 34% and 36%, respectively (p < 0.001). The median of CD4+ T cell counts among PALOP was lower 217cells/μL than Brazilian 370 cells/μL and Portuguese 289 cells/μL (p < 0.001) (We split migrants into two distinct groups: Portuguese speaking African Countries- PALOP and Brazil) and compared it with native population from Portugal. Based on that, migrants from PALOP are older (41yo) than Portuguese (39yo) and Brazilians 34 yo (< 0.001) .A total of 5177 samples were successfully screened for subtype analysis. Viruses were predominantly B , followed by subtype G , Recombinants , CRF02_AG , subtype C , subtype F1 , subtype A , subtype D  and others .p < 0.001) , followed by URFs (18%), subtype G (17%), subtype C (16.4%), subtype B (8.4%), A (5.5%) and other subtypes (8%). Within migrants from Brazil the most prevalent subtype was B (58.4%), followed by C (12%), recombinants (10%), G (8.6%), F1 (5.7%), and other subtypes (5%). The most prevalent subtype detect among Portuguese patients was B (44%) followed by subtype G (31%), URFs (11.2%), CRF02_AG (4%), subtype C (3.4%) and other subtypes (3.1%). Significant differences between PALOP, Brazil and Portuguese were observed (< 0.001) .p < 0.001) p = 0.161).Among the entire DN population of the study, at least one SDRM was detected in 342 out of the 3552 cases  in the HIV-1 polymerase. Mutations associated with NNRTI resistance, present in 186 cases , were the most common, followed by mutations associated with NRTI resistance 144 cases  and PI resistance 103 cases  . Within n = 59) harbored a singleton mutation . We detected 28% of primary resistance for dual distinct classes of drugs (NRTI/NNRTI) n = 26.Among migrants the majority (63.4%) of the TDR (p = 0.005). The logarithmic median of viral load in TDR group were lower (p = 0.001) and the median CD4 cell count were slightly higher than those in without TDR group (p = 0.024).Comparisons between patients infected with HIV-1 strains harboring any drug-resistant mutations and those without are shown in p = 0.007, p = 0.019, p = 0.015, p = 0.571, trend = 0.007.The trend of global TDR , M41L , T215F/Y/S . For NNRTIs the most prevalent mutations were K103N , Y181C/I , G190S/A  . For PIs, the most prevalent mutation was L90M  Overall, among the 144 DN patients with TDR to NRTI , associated with low to high resistance to a drug according to the Stanford HIV database algorithm v 8.4, the most prevalent mutations  were M184V  had mutations interpreted as moderate and high-level resistance. From those, 6.2% had moderate and high resistance to Efavirenz (EFV) and 2.8% to Rilpivirine (RPV). For NRTI, 82 sequences 8.06% had moderate and High-level resistance, 2.7% resistance to Emtricitabine (FTC), 2.6% to Lamivudine (3TC), 1.6% to abacavir (ABC) and 1.08% to Tenofovir (TDF). For PIs, we did not detect any moderate or High-level resistance .Among treated patients, the global ADR was detected in 980 out of the 1589 cases (61.6%). Within migrants, the overall prevalence of ADR was 61.5%, similarly to what was found in the autochthonous population (Portuguese patients) (62%) . NRTIs rp = 0.005, p <0.001, p <0.001, p = 0.841, The trend of global ADR  and TAMs, such as T215YF (18%). K103NS (24.9%) and L90M (5.7%) were the most prevalent mutations conferring resistance to NNRTIs and to PIs, respectively .To explore the predictors of transmitted and acquired drug resistance, we performed multivariable unadjusted and adjusted analysis using age, gender, CD4 count, VL at sample collection, subtype, country of origin, and sampling year. After adjustment, migrants from Mozambique were 1.7 times as likely to present TDR than migrants from PALOP and Brazil. Migrants infected with subtype B had 71% higher chance of harboring TDR. Patients sampled more recently were more likely to present TDR. The same analysis was done for ADR, and after adjustment, age, infection with subtype B, VL (log) and sampling year remained independently associated with ADR .In this study, we analyze the prevalence and trends of TDR and ADR in migrants followed up in Portugal between 2001 and 2017. With a sample size of 1490 migrants and 3687 autochthonous, we could analyze and compare the long-term time trend of TDR/ADR in these populations.Our population included 1281 (85.9%) migrants from Portuguese speaking African countries and 209 (14.1%) from Brazil. Those countries have long term important relations with Portugal and, except for Brazil, there is very little knowledge in HIV molecular epidemiology and surveillance of drug resistance mutations locally.Interestingly, the population of PALOPs had a median CD4 count much lower than that of Brazilian migrants and Portuguese population in general, indicating that these immigrants are diagnosed later. Another study conducted in Sweden with African migrants and the Swedish population found that among migrants the CD4 levels were significantly below the general population levels . AnotherA total of 7 subtypes, such as: A, B, C, D, F1, G, H, including CRF02_AG, UFRs, were detected among migrants and the identified subtypes were highly consistent with the ones causing epidemic in their countries of origin ,22,23. CWe observed a TDR rate of 9.1% among the 1017 antiretroviral-naïve PALOP and Brazilian migrants attending Portuguese HIV/AIDS clinics, low to moderate drug-resistance (5%–15%) according to the World Health Organization. This rate is very similar to that found in other sub-Saharan countries, including the neighboring Cameroon with a TDR around 9% . HoweverAs reported recently by CASCADE collaboration in Europe , the preAmong migrants from different Portuguese-speaking countries, migrants from Mozambique showed a high resistance rate of 17.4%, compared to the average of other Portuguese-speaking countries analyzed herein (7–10%). These results are discordant with other studies involving patients from Mozambique between the years 2007 and 2009 that showed a low prevalence of 5% of primary drug resistance based on WHO mutation list ,38. ThesWhile examining the presence of resistance by drug class, we found that there was a significant difference in the prevalence of drug-resistant mutations between the three antiretroviral classes  These SDRMs had an impact on the efficacy of several treatment strategies, especially efavirenz (EFV) and lamivudine (3TC), respectively. Between 2010 and 2017, EFV and 3TC were recommended for first line treatment in Portugal. This was also the most frequent NRTI and NNRTI-resistance mutation (M184V and K103N) among pre-treated patients and was previously reported among antiretroviral-naïve patients living in Portuguese Speaking countries ,40,41. AIn treated migrant patients followed up in Portugal, we found that the rate of acquired drug resistance was 61.5%. This high rate was coincident with the rate of ADR among autochthonous Portuguese patients (62%). Other European studies have revealed a high rate of ADR around 64% ,43. ThisInfection with subtype B was shown to be significantly associated with a higher risk of ADR in the univariate and multivariate analysis, as well as age and viral loads.n = 121). As such, and although TDR in migrants from Mozambique was found to be higher than that observed in the other migrants it is important to conduct other studies with a larger number of patients to better understand this issue.Our study has a number of weaknesses and limitations. Missing information about risk group limits interpretations about the importance of risk groups for transmission and acquisition of drug resistance. For example, the higher rate of DR found in subtype B infected patients could be associated with the higher proportion of men who have sex with men infected with this subtype . AnotherThis work highlights the importance of characterizing the ARV drug resistance profile in migrants. Through this report, it will be possible to better understand how HIV drug resistance is circulating in migrant populations and thus develop new strategies to better manage the epidemic.


Kaposi sarcoma herpesvirus (KSHV) is associated with Kaposi sarcoma (KS), primary effusion lymphoma, and multicentric Castleman disease (KSHV-MCD) in patients infected with human immunodeficiency virus (HIV). We present a case consistent with a newly recognized KSHV inflammatory cytokine syndrome (KICS), distinct from KSHV-MCD. Although both disorders exhibit signs of substantial inflammation, KICS has minimal lymphadenopathy/splenomegaly and negative pathologic nodal changes in the setting of low CD4 count. KICS is easily misdiagnosed as severe sepsis or other KS-related diseases in HIV/AIDS patients and carries a high mortality. Standard therapy is still under investigation due to its rarity, whereas the treatment regimen for KSHV-MCD may lead to clinical remission. Early recognition and prompt management are crucial to improve the survival of the under-recognized KICS. Kaposi sarcoma herpesvirus (KSHV), a human gamma herpesvirus 8 (HHV8), is associated with Kaposi sarcoma (KS), primary effusion lymphoma (PEL), hemophagocytic lymphohistiocytosis (HLH), and multicentric Castleman disease (KSHV-MCD) in patients infected with human immunodeficiency virus (HIV) -3. As a The lytic phase of HHV8 is characterized by massive stimulation and production of viral proteins, including viral IL-6 (vIL-6). As a homolog of human IL-6 (hIL-6), vIL-6 has similar biologic functions and structural characteristics. Although less potent, vIL-6 recruits macrophages and neutrophils, suppresses regulatory T-cell function, and induces tissue damage and hepatic acute phase response, by binding to the ubiquitous gp-130 transmembrane protein in human cells. Thus, it can still exert its effects despite bypassing the actual IL-6 receptor . MoreoveBecause of its rarity, KICS is easily misdiagnosed as severe sepsis or other KS-related disorders in HIV/AIDS patients and is, therefore, probably under-recognized . KICS syA 33-year-old African American male with a prior medical history of syphilis, HIV/AIDS on antiretroviral therapy (ART), and stage IV KS on doxorubicin was admitted due to worsening fatigue, bilateral leg swelling, and hyponatremia. He also had multiple complaints, including headaches, severe muscle and body aches, abdominal pain, nausea, vomiting, altered bowel habits, and shortness of breath. His chronic lower extremity edema had been getting worse with extension to his scrotum, he gained 20 pounds in two weeks before admission, and was unable to ambulate. His HIV/AIDS had been treated with Triumeq for more than one year, and his HIV VL was less than 20 copies/ml. However, his CD4 count was still low at 58 cell/µl not long before admission. Last doxorubicin for his KS was two months before. He did have mild lymphadenopathy at that time, while a left supraclavicular lymph node biopsy did not show any lymphoproliferative process or coexistent MCD. A repeat CT scan of his chest, abdomen, and pelvis upon current admission did not show any enlarged lymph nodes or hepatosplenomegaly except small bilateral pleural effusions and anasarca Figures , 2.Upon presentation, he was febrile at 102.6°F and chronically ill-appearing with dry mucous membranes. He had a distended abdomen as well as significant scrotal swelling in addition to 2+ pitting edema on his bilateral lower extremities. A small condyloma was noticed near his gluteal fold. Hyperpigmented lesions consistent with KS were noted all over his body, more significant on the lower extremities. His initial pertinent labs showed a white blood cell (WBC) count of 6 K/µl, sodium 122 mmol/L, albumin 1.8 g/dl, and lactic acid 1.7 mmol/L. Additional workups demonstrated negative urinalysis and no pneumonia on chest x-ray (CXR), although a left basilar opacity was consistent with a small pleural effusion Figure . AbdominThe patient's HIV treatment with Triumeq and antibiotic prophylaxis with azithromycin and trimethoprim-sulfamethoxazole were continued. While initially at baseline, his liver enzymes started trending upwards and he continued to have GI symptoms. A colonoscopy with biopsy of his rectal lesion was performed, which showed condyloma acuminatum with low-grade and focal intermediate grade anal intraepithelial neoplasia and concurrent ulcer with herpes virocytes. His HIV profiles were repeated, and his CD4 count remained low at 33 cell/µl with mildly increased HIV-1 viral load at 222 copies/ml (Table The patient had persistent hyponatremia (121-127 mmol/L) with low urine sodium, and did not respond to dose titration of tolvaptan and sodium tablets. His hypoalbuminemia was worsening despite an albumin infusion. His kidney function deteriorated during his hospital course, leading to acute kidney injury with electrolyte abnormalities. In addition, he developed severe anemia with a hemoglobin of 5.7 g/dl and thrombocytopenia of 47,000/μl (Table His significantly increased C-reactive protein 17.9 mg/dl) and the absence of lymphadenopathy/splenomegaly on CT images led to the suspicion of KICS. KICS was further confirmed by profoundly elevated IL-6 at 52 pg/ml, IL-10 at 2,600 pg/ml, and HHV8 VL at 14,855 copies/ml  . ComprehThe prognosis of KICS is poor compared to KSHV-MCD with a high mortality rate at 60%, is often refractory to available treatment, and carries a greater relapse rate after a short remission period ,5. The mAs a rare and distinct syndrome, KICS can often be confused with severe sepsis and carries a high mortality. Interestingly, unlike other opportunistic infections, it can persist despite good control of HIV viremia with ART. Early identification and immediate treatment initiation are crucial to improve survival of this under-recognized KSHV-associated disease. It is therefore imperative that physicians are aware and familiar with KICS, so that the diagnosis of KICS will not be missed or delayed when patients present with systemic signs of inflammation and have concurrent advanced HIV/AIDS along with KS. Moreover, current ongoing clinical trials and research may shed more light on this newly recognized entity by targeting the overproduction of vIL-6/IL-10 and dysregulation of systemic inflammation.


To reduce risk of antiretroviral resistance when stopping efavirenz (EFV)-based antiretroviral treatment (ART), staggered discontinuation of antiretrovirals (an NRTI tail) is recommended. However, no data directly support this recommendation.We evaluated the prevalence of HIV drug resistance mutations in pregnant women living with HIV who stopped efavirenz (EFV)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF) postpartum.3, initiated EFV/FTC/TDF in pregnancy and stopped ART at 6 weeks postpartum if formula feeding, or 6 weeks after weaning. A 7-day tail of FTC/TDF was recommended per Botswana guidelines. HIV-1 RNA and genotypic resistance testing (bulk sequencing) were performed on samples obtained 4–6 weeks after stopping EFV. Stanford HIV Drug Resistance Database was used to identify major mutations.In accordance with the prevailing Botswana HIV guidelines at the time, women with pre-treatment CD4 > 350 cells/mm3. The median time from cessation of EFV to sample draw for genotyping was 5 weeks (range: 3–13 weeks). Thirty-two (43%) women received a 1-week tail of FTC/TDF after stopping EFV. HIV-1 RNA was available from delivery in 70 (95%) women, 58 (83%) of whom had undetectable delivery HIV-1 RNA (< 40 copies/mL). HIV-1 RNA was available for 71 women at the time of genotyping, 45 (63%) of whom had HIV-1 RNA < 40 copies/mL. Thirty-five (47%) of 74 samples yielded a genotype result, and four (11%) had a major drug resistance mutation: two with K103N and two with V106M. All four resistance mutations occurred among women who did not receive an FTC/TDF tail , whereas no mutations occurred among 18 genotyped women who had received a 1-week FTC/TDF tail (p = 0.053).From April 2014 to May 2015, 74 women who had stopped EFV/FTC/TDF enrolled, with median nadir CD4 of 571 cells/mmViral rebound was slow following cessation of EFV/FTC/TDF in the postpartum period. Use of an FTC/TDF tail after stopping EFV was associated with the lower prevalence of subsequent NNRTI drug resistance mutation. Of note, pharmacokinetic variability (linked genetic factor – CYP2B6) in EFV metabolism between individuals has been observed, and EFV levels may be higher in persons of African descent.4 These higher drug levels may also impact risk of drug resistance when stopping EFV-based ART.Cessation of antiretrovirals raises concerns about the potential for the development of drug-resistant viral strains, particularly for non-nucleoside reverse transcriptase inhibitors (NNRTIs). Non-nucleoside reverse transcriptase inhibitors, such as efavirenz (EFV), have long half-lives and a low barrier to the development of drug resistance. When used in combination with other classes of drugs such as nucleoside reverse transcriptase inhibitors (NRTIs), staggered discontinuation using a 4–7-day ‘tail’ of the NRTI backbone when stopping NNRTI-based antiretroviral treatment (ART) is advised.3 without a WHO stage 3 or 4 illness take three-drug ART in pregnancy, but discontinue the ART postpartum or upon breastfeeding cessation (option B). Although both WHO and Botswana subsequently modified guidelines to recommend lifelong ART regardless of CD4 cell count or disease stage (option B+), the prior guidance for pregnant women, as well as the inconsistent application of an NRTI tail at the time of EFV cessation, offers an opportunity to evaluate the development of resistance and the performance of a TDF/FTC tail among women stopping an EFV/TDF/FTC regimen. Because this ART regimen remains widely used throughout the world, the natural experiment afforded by prior PMTCT guidelines is applicable to our understanding of the risk of cessation of NNRTI-based regimens generally.Prior to 2015, both the World Health Organization (WHO) and the Botswana programme for the prevention of mother-to-child transmission (PMTCT) recommended that pregnant women living with HIV with a CD4 cell count > 350 cells/mm5Between April 2014 and May 2015, we enrolled a subset of pregnant women living with HIV participating in a trial of infant cotrimoxazole prophylaxis in Botswana (the ‘Mpepu’ study) into this resistance sub-study. Mpepu was a double-blinded randomised controlled trial designed to assess the efficacy and safety of infant cotrimoxazole prophylaxis versus placebo used daily from as early as 14 days of life through 15 months among HIV-exposed uninfected infants in Botswana.3 without evidence of WHO stage 3 or 4 disease, initiated EFV/FTC/TDF in pregnancy and stopped ART postpartum according to Botswana guidelines at the time . Per Botswana national HIV treatment guidelines, a 7-day tail of TDF/FTC after cessation of EFV was recommended. However, decisions regarding receipt and cessation of maternal ART occurred at government clinics (rather than study clinics), and the 7-day tail was inconsistently applied at the time of ART cessation.Women were eligible for this sub-study of drug resistance following cessation of EFV/FTC/TDF if they were living with HIV, had pre-treatment CD4 > 350 cells/mmWomen eligible to participate in this sub-study were identified in the Mpepu study and scheduled for a clinic visit 4–6 weeks after ART cessation. Interested and eligible women consented to participate in the study and provided data  on PMTCT regimen received; dates on which ART and individual antiretrovirals were started and stopped as well as provided blood specimens for drug resistance and HIV-1 RNA testing. HIV RNA results at the point of enrolment into the Mpepu study, and documented nadir CD4 counts, were retrieved and served as baseline values.6 We then performed reverse transcription using in-house one-step RT-PCR technique.7 Polymerase chain reaction (PCR) products were then purified8 and sequenced.9 Consensus sequences were generated, and the Stanford HIV Drug Resistance Database was used to identify all drug resistance mutations in the protease and reverse transcriptase coding regions.Prior to drug resistance testing, we first determined HIV-1 RNA viral load levels with the Abbott m2000sp/rt machine (limit of detection 40 copies/mL). Where HIV-1 RNA was detected, it was extracted using an automated validated technique.Statistical Package for Social Sciences (SPSS) version 25 was used to perform statistical analyses, which were generally descriptive in nature; a two-sided Fisher’s exact test was used to evaluate for differences in the proportion of drug resistance by the approach to ART cessation, either with a 7-day TDF/FTC tail or with abrupt cessation of all three drugs. The small number of women with drug resistance mutations precluded more detailed analysis of predictors of drug resistance.The Botswana Health Research Development Committee and the Office of Human Research Administration at Harvard T. H. Chan School of Public Health approved this drug resistance sub-study (HRDC 00732 and IRB13-2772), and women provided written informed consent for participation.3 post-delivery and had to continue ART for their own health per Botswana national protocol at the time. The median time from cessation of EFV (whether or not a tail period occurred) to time sample was collected for resistance testing was 5 weeks (range: 3–13 weeks). Median age at enrolment was 29 years (range: 20–45) with median nadir CD4 count of 571 cells/mm3 (range: 361–1236). Forty-seven (64%) women had no previous exposure to antiretrovirals, 25 (34%) reported previous exposure to antiretrovirals for PMTCT purposes in a prior pregnancy and 2 (2%) were previously exposed to EFV/FTC/TDF but stopped prior to conception and then re-started EFV/FTC/TDF as three-drug prophylaxis for the index pregnancy. Thirty-two (43%) stopped ART with the recommended 1-week tail of TDF/FTC after cessation of EFV and 42 (57%) stopped all treatment at once. Of 70 women with viral load results at enrolment into the Mpepu study, 58 (83%) had HIV-1 RNA < 40 copies/mL. At 4–6 weeks post-EFV/FTC/TDF cessation, 44 (62%) of 71 women with viral load result had HIV-1 RNA < 40 copies/mL. Thirty-three (47%) women had HIV-1 RNA < 40 copies/mL at delivery and at the time of sample draw for genotyping (post-ARV cessation).Ninety women enrolled, 74 of whom discontinued EFV/FTC/TDF postpartum and had samples collected for genetic resistance testing and are included in this analysis. Sixteen of 90 women not included in this analysis had CD4 < 350 cell/mmp = 0.053). Eighteen (58%) of the 31 women who had a genotype result and no drug resistance mutation took a TDF/FTC tail. Of 39 samples that could not be sequenced, 37 (95%) had HIV-1 RNA < 40 copies/mL in the sample being tested.Thirty-five (47%) of 74 samples were successfully sequenced, with 4 (11%) of 35 having a major drug resistance mutation: 2 with K103N and 2 with V106M . All fouA small proportion of women stopping EFV/FTC/TDF had a major NNRTI resistance mutation detected in plasma taken a little more than a month after stopping treatment. Among women receiving a 7-day tail of TDF/FTC after stopping EFV where genetic resistance testing was able to be performed, none had detectable mutations. Our findings thus support the recommended staggered discontinuation of antiretrovirals when an EFV/FTC/TDF regimen is being stopped. Our data support prior studies that evaluated the use of an NRTI tail when discontinuing EFV-based ART.10 We believe this could be because of low pre-ART viral loads in our population (because of women with less advanced disease starting ART in pregnancy).Our observation of prolonged viral suppression < 40 copies/mL for a median of 5 weeks in 62% of women post-ART cessation was similar to findings from other studies which have demonstrated that it is not unusual for viral suppression to persist for weeks after stopping ART.Our study had several limitations. Firstly, our results are based on a small number of observations making generalisability challenging. Secondly, we did not report on minor drug resistance variants and, finally, more than half (63%) of women tested at a median time of 5 weeks from EFV cessation were still virally suppressed, which may suggest that we might have tested for resistance strain development early. Thirdly, we could not determine why the 7-day tail was inconsistently applied at the time of ART cessation and could therefore have missed evaluating possible confounders related to this factor.Although guidelines no longer recommend discontinuation of ART after pregnancy, our study has general applicability for those requiring permanent or temporary treatment discontinuation when receiving an EFV-based regimen, informing the risk of mutation emergence in the setting of abrupt three-drug discontinuation versus discontinuing with a TDF/FTC tail. Furthermore, despite the shift to the use of newer drugs such as dolutegravir for which there is less concern about the emergence of resistance than with NNRTIs, our data are relevant to millions of individuals still on EFV-based ART .Based upon our results, we conclude that an TDF/FTC tail may help prevent selection of major NNRTI drug resistance mutations that can occur with the abrupt discontinuation of an EFV/FTC/TDF ART regimen.


While the proportion of HIV-positive children (under 15 years) enrolled on antiretroviral therapy (ART) has increased in recent years, up to 60% of children started on ART do not achieve virological suppression. We set out to determine the factors associated with virological non-suppression among children living with HIV receiving ART at a peri-urban HIV care clinic in Kampala, Uganda.This was a retrospective cohort study conducted at the pediatric HIV/AIDS clinic at the Joint Clinical Research Centre (JCRC) in Kampala, Uganda. Three hundred (300) HIV-positive children (0–14 years) were randomly selected from existing medical records and data on children’s socio-demographic and clinical characteristics  were abstracted using a data abstraction form. Virological non-suppression was defined as a viral load ≥1000 copies/Ml of blood after six months of ART initiation. Incident rate ratios (IRRs) were determined as a measure of association between virological non-suppression and child/patient characteristics. The IRRs were obtained via a modified Poisson regression with corresponding 95% confidence intervals (95%CI). All analyses were done using statistical package, Stata version 15.The overall non-suppression rate among HIV-positive children on ART was 23%. Being at WHO clinical stage 4 at ART initiation  and ART-induced side effects  were significantly associated with non-suppression. Older age at ART initiation (age 5–9 years: ; age 10–14 years:  was less likely to be associated with virological non-suppression.Nearly a quarter of HIV-positive children on ART had a non-suppressed viral load after six months of treatment. Being at WHO clinical stage 4 at ART initiation and ART-induced side effects were significantly associated with virological non-suppression while older age at ART initiation was protective. Our findings suggest a need for age-specific interventions, particularly those targeting children below five years of age, to improve virological suppression among HIV-positive children receiving ART in this setting. In East and Southern Africa out of the 1,400,000 HIV-positive children, only 51% are on antiretroviral therapy (ART) , the supIn low and middle income countries, a viral load (VL)<1000 copies/ml defines treatment success (suppression), a measure of ART efficacy, which also indicates treatment adherence and reduced risk of HIV transmission . SeveralIn Uganda, regular VL monitoring is done at the Central Public Health Laboratory through the districts’ laboratory hubs and in case of virological non-suppression, that is; two consecutive viral loads above 1000 copies/ml done at least 3–6 months apart , 3 sessiIn Uganda, approximately 95,000 children are living with HIV, 47% of them receiving ART, with a non-suppression rate of 60.1%, which is higher than that observed among adults (40.4%) and also far above the national target of 10% . AlthougThis study was conducted at the Joint Clinical Research Centre (JCRC), hereafter referred to as ‘the Centre’, a medical research institution in Uganda specializing in HIV treatment and management . The CenThe study used a retrospective cohort study design through the reviews of medical records to establish the child/patient factors associated with virological non-suppression among children (0–14 years) living with HIV receiving ART.The study population was HIV-positive children aged 0–14 years receiving ART in the pediatric HIV clinic at the Centre, enrolled between January 2017 and March 2019. Only children who had been on treatment for at least 6 months and had VL test results taken between 6 and 8 months after initiating ART, were considered for this analysis.The sample size for determining the proportion of children living with HIV who had a non-suppressed viral load after six months of receiving ART was estimated using the Kish Leslie (1964) formula, assuming a 95% Confidence Interval, an estimated prevalence of virological non-suppression among HIV-positive children on ART of 60.1% based on the Uganda population-based HIV-impact assessment report 2017 , and a mThe children included in the analysis were selected from the pediatric HIV database using systematic random sampling. We used clinic records to generate a sampling frame comprising patients’ serial numbers that were assigned to children’s files at the time of admission into the HIV clinic. With an estimated sample size of 300 and a population size of 1,019, we determined a sampling interview of three (3), suggesting that every third child on the sampling frame was selected to participate in the study. The first participant lied between the first and the third participant and was selected using simple random sampling, ballot method without replacement.A semi-structured data abstraction form was designed by the first author (SN) and used to extract demographic , and clinical  child/patient-related factors from the HIV care database. We employed research assistants who were nurses working at the Centre to help with the data abstraction. We developed a detailed manual of operations and standard operating procedures to guide data collectors on how to abstract the data from the database, with guidance from the first author. The data abstraction tool was pre-tested at the Centre a week prior to the start of the study. During pre-testing, data for 20 HIV-positive children on ART were abstracted from the pediatric HIV database by the Research Assistants, working closely with a Data Manager. These data were assessed for completeness and accuracy by the first author who also validated each question and the possible responses and refined the tool based on the pre-testing feedback received from the Research Assistants, accordingly. A week later, the study team started full-scale data abstraction to obtain the records for 300 HIV-positive children on ART as needed. The abstraction exercise was monitored frequently to ensure that the process was implemented as expected. At the end of each day of data abstraction, the first author reviewed the data collection forms to ensure completeness and accuracy of records. Incomplete forms were returned to the data collectors to repeat the data abstraction exercise until all essential records were retrieved. We further kept track of the data flow process to avoid loss of data forms, assessed the performance of each research assistant and met with research team regularly to discuss progress or any problems that cropped-up.yes’—having registered any side-effect or ‘no’—having not registered any and ART regimen was categorized as Nevirapine (NVP)-based, Efavirenz (EFV)-based or Lopinavir/ritonavir(LPVr)-based regimens.The primary outcome variable for this study was virological non-suppression after six months following initiation of ART, defined as viral load of at least 1000 copies/ml of blood. Age at initiation in completed years was categorized in to 3 sub-categories: , sex was categorized as either male or female, TB-co-infection was measured as currently receiving TB treatment or within the past one year, WHO clinical staging at ART initiation included all the stages; i.e. stages I-IV; adherence level was determined by pill-count/self-reports and sub-categorized as ‘poor’  or ‘high’ adherence (95%-100%), based on the 2018 Ministry of Health’s consolidated guidelines for the prevention and treatment of HIV and AIDS in Uganda Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Reviewer’s ReportThe aim of this research paper was to identify the possible underlying factors associated with virological non-suppression among children living with HIV on ART. This study was based on previous ones done in other centres which showed high rates of virological non-suppression among HIV children on ART. The authors went forward to establish those factors at their pediatric HIV/AIDS clinic. Purportedly, the study results would be based on, to put in place measures that would improve the treatment outcomes of HIV infected children in their health facility.The study was well designed, had a reasonable number of participants and used a good analysis technique. The tables are easily understood and match the information in the text. The conclusion made is realistic and in keeping with the study findings.The authors found that the virological non-suppression was at 23%. Being at WHO clinical stage 4 at ART initiation of ARVs and having ARV-induced side effects during treatment were the identified risk factors significantly associated with virological non-suppression.Basing on the above observations, this paper would be a good basis for further studies on the same topic and worth publishing in an international journal like PLOS ONE.However, as a retrospective study, it has its own shortcomings. The possible limitations of this research were elaborated on in their discussion chapter and are understandable.There are minor issues which the authors should clarify on:1. Being at WHO clinical stage 4 at ART initiation per se is a risk factor for virological non-suppression, however, there could be other underlying confounding factors common in stage 4 HIV disease which need to be highlighted with examples in the discussion. This will help clinicians to identify and manage them so as to improve the treatment outcomes2. The authors should comment on the findings that “Non-suppression was higher in children with adherence level of 61-80% , than in those with an adherence level of 40-60%  and 81-100% ”. Page 16, 2nd last sentence. It is well known and expected that poor compliance to ARTs is associated with virological non-suppression.I a nutshell, this is a research paper that is worth publishing in PLOS ONEReferenceshttp://www.ijpediatrics.com1. Ravichandra KR et al. Int J Contemp Pediatr. 2017 Sep;4(5):1743-1747 Reviewer #2: General commentsThis is an important toward in the light of countries efforts towards the attainment of the UNAIDS 90 90 90 or 95 95 95 targets by 2030. In addition, paediatric HIV is heavy burden mainly in sub Saharan Africa. The challenges with diagnosis, and more importantly managing paediatric HIV makes this paper an important one. While many countries have made significant progress in managing HIV among adults with resultant good virological suppression rate, this has proven to be very challenging for the paediatric population.Suggestions for improvement1. The manuscript has no page nor line numbering making review difficult2. There are still some typographical and grammatical errors, kindly read through again and edit3. Ethical clearance: state the ethical clearance reference numbers4. In the abstract methods, you use =>, this is not the standard symbol used. Please use ≥5. In this study you only focus on suppression rate at 6 months. What was the 12 months? Since this was a retrospective data collection, why was this not reported?6. Can you state that truly all the 300 children had exactly 6mths on ART viral load results available? Or were some taken between 6-9mths etc. as is commonly the case?7. In the background, second paragraph, you have this long and difficult to follow sentence “Several factors like; low adherence rate [4], WHO clinical stage 4 and TB co-infection have been highlighted to be associated with virological non-suppression among adults [5], and viral load suppression rates among children on ART have been [1] shown to be low and considerably poorer [6],[7].” Try to break this into 2 sentences to improve clarity.8. In the methods section under measures, you categorised level of adherence as “Poor 40%- 60%, Medium 61%-80% and High 80% -100%)”. What informed this? My main concern is 80% being considered as high adherence, so explain this and let readers understand why this.9. Results, table 2: please state the actual p-values10. Discussion, second paragraph; you compare your rate of viral non-suppression to some countries findings without giving any critic or comparison of the study populations. Example, were the ages similar as in this study and any other factors which could have modified their findings compared with this study?11. What is the best study design which would have better given a true picture of the factors affection suppression rate? You stated some limitations of this study and I was therefore expecting some thoughts on example what a prospective study could contribute better.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  10 Jan 2021Point-by-Point Response to Academic Editor and Reviewers’ Comments1. Academic Editor’s commentsS/N Comments Response1 Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming Thank you for the polite reminder, yes we have ensured that the manuscript meets all the PLOS ONE style requirements.2 Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information. We developed a data abstraction form in English as part of the study, that was used by the research assistants (nurses). We have provided a copy of this form, in English language, as supporting information.3 You have mentioned that the questionnaire was pre-tested. Please clarify if it was how it was pretested and validated. Yes, we have provided clarity on how the data abstraction tool was pretested and validated under the data extraction procedures on page 6, as;The data abstraction tool was pre-tested at the Centre a week prior to the start of the study. During pre-testing, data for 20 HIV-positive children on ART were extracted from the pediatric HIV database by the Research Assistants, working closely with a Data Manager. These data were assessed for completeness and accuracy by the first author who also validated each question and the possible responses and refined the tool based on the pre-testing feedback received from the Research Assistants, accordingly. A week later, the study team started full-scale data extraction to obtain the records for 300 HIV-positive children on ART as needed. The extraction exercise was monitored frequently to ensure that the process was implemented as expected.4 We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions. In your revised cover letter, please address the following prompts: a). If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail  and who has imposed them . Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. There are no ethical or legal restrictions on sharing a de-identified data set, and this has been clearly stated in the rebuttal letter. b). If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. The data set necessary to replicate our study findings has been uploaded to the PLOS ONE. 5 Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section. The ethics statement appears only in the methods section.2. Reviewer #1’s commentsS/N Comments Response1 The study was well designed, had a reasonable number of participants and used a good analysis technique. The tables are easily understood and match the information in the text. The conclusion made is realistic and in keeping with the study findings. Basing on the above observations, this paper would be a good basis for further studies on the same topic and worth publishing in an international journal like PLOS ONE. We thank the reviewer for these compliments. We agree and hope that our paper will serve as a basis for further studies on virological non-suppression among HIV-positive children on ART, as we have recommended.2 Being at WHO clinical stage 4 at ART initiation per se is a risk factor for virological non-suppression, however, there could be other underlying confounding factors common in stage 4 HIV disease which need to be highlighted with examples in the discussion. This will help clinicians to identify and manage them so as to improve the treatment outcomes Thank you for pointing this out, we had missed indicating it. We have now addressed this concern page 13, as:This is likely because such children have advanced HIV-infection with severe symptoms like a very low CD4 cell count as well as rapid disease progression, with Aids-defining illnesses like pneumocystis pneumonia, toxoplasmosis and ctyomegalo infections among others. All these illnesses need to be identified and attended to by clinicians for the children to achieve suppression, since they e halt the effectiveness of ART-regimens3 The authors should comment on the findings that “Non-suppression was higher in children with adherence level of 61-80% , than in those with an adherence level of 40-60%  and 81-100% ”. Page 16, 2nd last sentence. It is well known and expected that poor compliance to ARTs is associated with virological non-suppression. We have also addressed this concern and commented on this issue in the discussion section: second last paragraph, on page 15, as; Although it is well known that poor adherence is associated with non-suppression, the proportion of children with non-suppressed viral loads after 6months of treatment was higher in children with adherence level of 85-94% than in those with an adherence level of 40-84% and 951-100%. However, this was not statistically significant and the frequency counts of <5 could explain this controverting finding. Besides, the adherence was self-reported and this too may have biased the findings .3. Reviewer #2’s commentsS/N Comments Responses1 This is an important toward in the light of countries efforts towards the attainment of the UNAIDS 90 90 90 or 95 95 95 targets by 2030. In addition, pediatric HIV is heavy burden mainly in sub Saharan Africa. The challenges with diagnosis, and more importantly managing pediatric HIV makes this paper an important one. While many countries have made significant progress in managing HIV among adults with resultant good virological suppression rate, this has proven to be very challenging for the pediatric population. Thank you so much for this feedback. We do hope that this paper will provide the basis for addressing pediatric HIV treatment challenges in sub-Saharan Africa in the efforts to achieve the 90-90-90/ 95-95-95 targets. 2 The manuscript has no page nor line numbering making review difficult. Thank you so much for raising this, it was an inadvertent error. We have page numbered the manuscript.3 There are still some typographical and grammatical errors, kindly read through again and edit Yes, we also realized as we read through again, that there were some typographical and grammatical errors. We have edited the manuscript, thank you for pointing this out.4 Ethical clearance: state the ethical clearance reference numbers This study was conducted for the MPH dissertation for the first author (SN). Makerere University School of Public Health’s Institutional Review Board, the body that cleared the research, doesn’t provide reference numbers for MPH student research studies. This explains why we did not include the ethical reference numbers. 5 In the abstract methods, you use =>, this is not the standard symbol used. Please use ≥ Thanks again for pointing out this error; we have corrected it. See page 7  and page 2 (Abstract: methods section) for details.6 In this study you only focus on suppression rate at 6 months. What was the 12 months? Since this was a retrospective data collection, why was this not reported? We recognize that we alluded to the 12 months in the background section, first paragraph on page3, stating; It is recommended that children with initial positive virological test results are initiated on ART immediately and routine viral load monitoring be carried out at 6 and 12 months, then every 12months if the patient’s viral load becomes stable.In stating so, we wanted to indicate the time points that are recommended for monitoring children on HIV as per the 2018 Uganda Ministry of Health consolidated guidelines for the prevention and treatment of HIV infection. However, we restricted our study to 6 months because it is expected that with effective treatment and good adherence, the patient must be able to achieve suppression after 6 months on treatment. 7 Can you state that truly all the 300 children had exactly 6mths on ART viral load results available? Or were some taken between 6-9mths etc. as is commonly the case? Not all the 300 children had VL testing at exactly 6 months. We included children in the study based on availability of VL test results between 6-8months of ART initiation. We had not made this clear before, but now, it is clearly stated under the methods section: study population page 5.8 In the background, second paragraph, you have this long and difficult to follow sentence “Several factors like; low adherence rate [4], WHO clinical stage 4 and TB co-infection have been highlighted to be associated with virological non-suppression among adults [5], and viral load suppression rates among children on ART have been [1] shown to be low and considerably poorer [6], [7].” Try to break this into 2 sentences to improve clarity. Thank you so much for this important observation. This very long sentence has now been broken into two sentences as shown on page 3, as:Several factors like; low adherence rate [4], WHO clinical stage 4 and TB co-infection have been highlighted to be associated with virological non-suppression among adults [5]. Likewise viral load suppression rates among children on ART have been shown to be low [1] and considerably poorer [6],[7].9 In the methods section under measures, you categorized level of adherence as “Poor 40%- 60%, Medium 61%-80% and High 80% -100%)”. What informed this? My main concern is 80% being considered as high adherence, so explain this and let readers understand why this. We have gone ahead to re-categorize the levels of adherence, in accordance with the 2018 Uganda Ministry of Health consolidated guidelines for the prevention and treatment of HIV infection, and this is now well-stated under ‘Measures’ within the methods section. This re-categorization has resulted in minor changes within the original findings but these changes did not affect the original interpretation and/or direction of results in any way. These minor changes appear on pages 2, 6, 8,9 ,10 and 11.10 Results, table 2: please state the actual p-values Thank you for pointing out this, the actual p-values are now stated on page 1011 Discussion, second paragraph; you compare your rate of viral non-suppression to some countries findings without giving any critic or comparison of the study populations. Example, were the ages similar as in this study and any other factors which could have modified their findings compared with this study? We now realize how important this is, thank you for bringing it to our attention. We have included a comparison of the findings and/or populations studied; see page 12. This is what we have included in the revised paper: For instance, a study conducted among HIV-positive children in rural Zambia found that only 11.5% of the children were virologically non-suppressed after six months of treatment [15]. Similarly, another study conducted in Zimbabwe found that 19% of the children were virologically non-suppressed, although the study was done in a hospital that specialized in managing opportunistic infections and managing complicated referrals of HIV patients on second and third line treatment, which could explain the lower proportion of non-suppression among the children. [16]. Nevertheless, all these studies were conducted in tertiary referral HIV treatment centres, and therefore, the higher proportion of children with non-suppressed viral loads after six months of treatment observed in our study indicates the need to enhance the strategies aimed at improving suppression among HIV-positive children especially in lower level health facilities, especially the public ones, since attending a public health facility has been shown to be associated with sub-optimal adherence in sub-Saharan Africa and Asia [17]. 12 What is the best study design which would have better given a true picture of the factors affecting suppression rate? You stated some limitations of this study and I was therefore expecting some thoughts on example what a prospective study could contribute better. We missed pointing out this yet it is vital. Thank you for raising this concern. We have stated the best study design that would have given a true picture on page 14, as:Although it requires more resources , a prospective study design would have been the best alternative to minimize selection bias and provide more clarity on the temporal sequence of the virological suppression and the above-mentioned associated factors. 14 Jan 2021Factors associated with Virological Non-Suppression among HIV-Positive Children Receiving Antiretroviral Therapy at the Joint Clinical Research Center in Lubowa, Kampala UgandaPONE-D-20-25133R1Dear Ms Nabukeera,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Professor Kwasi Torpey, MD PhD MPHAcademic EditorPLOS ONEAdditional Editor Comments :Comments addressedReviewers' comments: 18 Jan 2021PONE-D-20-25133R1 Factors Associated with Virological Non-Suppression among HIV-Positive Children Receiving Antiretroviral Therapy at the Joint Clinical Research Centre in Lubowa, Kampala Uganda Dear Dr. Nabukeera:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. 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