LPV/r) oral pellets were developed to overcome challenges with administration and storage experienced with previously available tablet and syrup forms of LPV/r prescribed to paediatric HIV patients. We report on the adoption of LPV/r pellets for infants living with HIV in the public sector antiretroviral therapy (ART) programme in Zimbabwe.Heat‐stable lopinavir/ritonavir (LPV/r‐based regimen (including ART‐naïve patients) in fourteen facilities across the country were eligible to receive the pellets. Caregivers were counselled on the new formulation and provided with administration guides. A caregiver questionnaire was administered three to four months after the child initiated on pellets. Data were also extracted from patient ART records.Infants aged three months to three years who had been prescribed a p = 0.013) times as likely to experience a challenge.One hundred and fifty‐seven children were enrolled . Survey data from 74 caregivers were included for analysis. Eighty‐one per cent of the caregivers preferred pellets while 19% preferred the syrup formulation. Eighty‐nine per cent assessed their child's response to taking the pellets as good or very good. Overall, 46% did not report any challenges while 54% reported one or more challenges with using the pellets. Difficulties with administration included: poor taste ; swallowing pellets ; finishing the dose ; and opening the capsule . Caregivers who were not confident to instruct others on pellet administration were 5.64 (95% confidence interval 1.45 to 21.95, LPV/r and reported a good response to pellets; however, they also reported challenges with administration. Counselling should focus on ensuring that caregivers can confidently administer pellets and are able to instruct others, to ensure high uptake and good adherence to treatment. LPV/r pellets may be an acceptable substitute for other available forms of LPV/r for eligible children under three years if they are currently on or in need of LPV/r‐containing regimens; however, challenges with administration still highlight the need for improved drug formulations for paediatric ART patients.A large proportion of caregivers preferred pellets to other formulations of  Available since 2015, the pellets represent an optimized paediatric ARV in Zimbabwe. Prior studies have shown the tolerability of pellets to be similar to that of the syrup 2A small, initial procurement of pellets was made in Zimbabwe prior to large‐scale rollout of the product, allowing sufficient medications for up to 200 children for a year. In order to ensure that learning from this first procurement could be applied during the later rollout, a pilot was established with the goal of enrolling 200 children living with HIV. Eligible children were enrolled according to the inclusion/exclusion criteria outlined in Table 2.1In the period February to July 2017, data were collected using questionnaires administered to caregivers by counsellors and/or nurses at routine visits and to health workers by the study team. Data were also collected from patient records regarding patient immunological status (CD4 percentage), viral load (VL) and weight‐for‐age Z‐score at the time of pellets initiation. For measures that were not available at the time of initiation, latest data from within three months of pellets initiation were used for current patients. For newly initiating patients, VL and CD4 tests were conducted at the time of initiation.2.2Data from the questionnaires were analysed with STATA 14.2, StataCorp LP  to assess for trends in administration methods and challenges. Factors associated with challenges were assessed using bivariate logistic regression. Measures of central tendency for immunological test results were also analysed for patient monitoring purposes.2.3Approval to undertake the study was granted by Chesapeake institutional review board on 19 April 2017 (Pro00018801). The study also had approval from the Medical Research Council of Zimbabwe and the Medicines Control Authority of Zimbabwe.3. The median age at the time of initiation on pellets was twenty‐one months . A child above three years (outside inclusion criteria) was allowed to enrol as they could not tolerate any other LPV/r formulation. At the time of pellet initiation, 11.4% (n = 18) of children were ART naïve. Among patients already on treatment, the median duration on ART at baseline was ten months . At baseline, 58 patients (40.6%) had a CD4 percentage measurement available (median: CD4% 25.5%) and 53 patients (37.1%) had a baseline VL reading (median: 188 copies/μL). Of 53 children on treatment at baseline with VL result (treated with LPV/r syrup for an average of five months), 19 (36%) had a VL above 1000 copies/mL at baseline while 17 (32%) had an undetectable VL (below 50 copies/mL).A total of 157 HIV‐positive children  were enrolled and initiated on the pellets. Urban facilities recruited 68% of the patientsThe caregiver experiences questionnaire Appendix  was compOver the period of the pilot, a total of 34 clients exited the study, representing a drop‐out rate of 21.6%. These patients had been prescribed the pellets for 2.6 months on average when they exited the study or were determined to be loss to follow‐up. Twenty‐five (73.5%) of the individuals who exited the study were taking the LPV/r solution at baseline and the rest were ART naïve and began treatment taking the oral pellets. Seven per cent of patients exited between weeks 3 and 9 of the study period. The main reasons for the patients withdrawing from the pilot were largely due to the child vomiting and not tolerating the pellets. Other reasons not related to the pellets for exiting are detailed in (Table Caregivers who completed the survey (n = 74) had been administering the pellets for an average of 3.2 months (SD 1.9 months). Forty‐six per cent (34 participants) reported no challenges with administering the pellets and the remaining 54% (40 participants) reported at least one challenge. The number of times each specific challenge was reported is detailed in . Furthermore, if a caregiver reported that the pellets were not working as well as the syrup, then they were also 7.7 times as likely to report a challenge with administration .An analysis was conducted to assess for factors that could be associated with having one or more administration challenges with the pellets. If a caregiver reported that they were not confident to instruct others on administration of the pellets, then they were 5.64 times as likely to report challenges with administration  1.45 to 21.95, Although the analysis does not establish any causal relationship between factors, the findings point to several questions that health workers could ask caregivers to screen for administration difficulties. Specifically, health workers could ask “How confident do you feel to instruct others on how to administer the pellets?” warranting additional support should the respondent report he/she is not very confident. This screening could be made more effective by timing it around the three‐week mark post pellet initiation, when more patients in this study were likely to abandon use of the drug.3.1With regard to how well the caregiver felt the pellets were working for their child, 66.7% (48 participants) of caregivers thought that it was better than the LPV/r syrup, 13.9% (10 participants) thought it was the same as the syrup and the remaining 19.4% (14 participants) did not think that the pellets were as good as the syrup. Furthermore, 53.5% (38 participants) reported that the child's response to the pellets was very good, while only 10.3% (eight participants) reported a poor or very poor response to the pellets.3.2Of the 53 patients with a recorded baseline VL test result, 14 had a second VL result at the end of the study period. Of the 19 patients, 12 with a baseline VL above 1000 copies/mL had a repeat VL taken at the end of the study period; eight had achieved viral suppression  after six months of using the pellets; four still had a VL above 1000 copies/mL.3.3The relatively high caregiver acceptability (81%) and preference demonstrated by the caregivers’ responses can be mainly attributed to the improved tolerability of the drug by the children, perhaps as function of the pellets’ improved palatability over the syrup formulation and the ability to easily co‐administer it with soft foods such as porridge. These findings are supported by various studies The study, however, demonstrated tolerability of the pellets to be an issue, with parents citing vomiting as a side effect in one in five children. This echoes findings from the CHAPAS2 study In the light of the importance of correct administration and improving tolerability, one of the key recommendations from this pilot is that the messaging on how to administer the pellets should be supported by practical demonstrations from healthcare workers or expert mothers in peer support groups to ensure correct practice. Additionally, caregivers should be asked to demonstrate their understanding of the instructions provided before leaving the facility. Complementary to this education on pellet administration, caregivers should also be given pamphlets that highlight the potential side effects of the drug and ways to address them. This would encourage caregivers to better report adverse events and seek assistance on addressing challenges affecting their infant's tolerance of the medication.et al. Giralt 4This pilot originally expected to enrol 200 patients based on the availability of medications and paediatric patient loads at the selected sites; however, only 78.5% of the expected number was enrolled. According to health worker feedback, the main reasons for low enrolment included: caregiver reticence in signing the required consent forms (but not necessarily having resistance to initiation of pellets themselves), and initial discomfort on the part of the health workers with both instructing caregivers on the pilot procedures and with initiating paediatric patients. This feedback indicates that these issues may have related to research barriers rather than problems with the pellets, and thus, pilot enrolment may not be indicative of expected pellet uptake outside of the context of a pilot study.Only 74 caregivers from the expected 157 responded to the survey. This pilot took place in public sector health facilities and questionnaires were administered by health workers in the facility who received no additional incentives from the study. While this realistic context provides insight into how pellets would be rolled out at scale, it also presented some challenges, which made it difficult to collect questionnaires from all the patients within the study time frames. In particular, patients returned for ART consultations on irregular schedules and health workers sometimes failed to complete the questionnaire.5This pilot supports that LPV/r oral pellets are a viable alternative formulation that patients and caregivers prefer for treatment of young children living with HIV. The results of this pilot support the substitution of pellets for other LPV/r formulation for children on ART in Zimbabwe's context and will inform the full scale rollout of this formulation nationally. The administration challenges noted should serve to guide further improvements in paediatric formulations beyond the oral pellets and contribute towards closing the gap between the optimal regimen options available to children and adults.The authors declare that they have no competing interests.All co‐authors contributed in designing the operational study. BP and RK oversaw the data collection performed during the study. OS, through Drugs for Neglected Diseases Initiative (DNDi), supported the provision of LPV/oral pellets administered in the study. MLP and BP analysed the data, and worked with RK, TA and BS to write the first draft of the paper. All co‐authors contributed to interpreting findings and writing the manuscript.


The WHO urges action against the threat posed by HIV drug resistance. It is well known that the sensitivity of Next-Generation Sequencing (NGS) is greater than that of Sanger Sequencing (SS). The objective of this study was to evaluate the performance of the novel NGS HIV-1 drug resistance monitoring system.Sentosa SQ HIV Genotyping Assay from Vela-Dx. This kit was used on a semi-automated Ion Torrent-based platform. Sequences were compared to those obtained by SS. Samples were analysed in the same and in separate runs. Quality controls (QC) were added to control sequencing processes of protease (PRO), reverse transcriptase (RT) and integrase (INT) regions.NGS analyses were performed on 67 plasma samples from HIV-1 infected patients using the Of the 41 analysed samples, 33 (80.5%) had identical drug resistance interpretation reports. Discrepant results were observed for eight samples. Five of them were only detected by NGS and had drug resistance mutations (DRMs) with an allelic frequency below the limit of detection of the SS method (between 6.3 to 20.5%). Two DRMs were only identified using the SS method. The sequences were similar in 98.2% of cases (counting variants as mismatches) and homologous in 99.9% if missed variants. Duplicated samples in a single run were similar in 95.7% (99.9%) of cases. Duplicated samples in two different runs were 98% (100%) homologous. QC results were manually assessed with a score of 340/340 for detection of DRMs in PRO and RT and 100% for INT sequencing.Sentosa SQ HIV Genotyping Assay. The NGS appears to be a promising tool for the detection of DRMs in HIV-1 patients and showed a higher sensitivity compared to SS. Large studies assessing the clinical relevance of low frequency DRMs are needed.This is the first preliminary evaluation in Belgium employing the  Human immunodeficiency virus (HIV) type 1 is the agent responsible for acquired immunodeficiency syndrome (AIDS) and for the majority of HIV infections worldwide . AlthougThe current treatment for HIV consists of a combination of antiretroviral drugs (ARV), typically three drugs from two or more classes . The ARVViral load is the most important indicator of initial and sustained response to ART and should be measured in all patients with HIV at entry into care, at initiation of therapy and on a regular basis thereafter. The level of viral load level before treatment is also an important factor in the selection of an initial ARV regimen because several currently approved ARV drugs have been associated with poor responses in patients with high baseline viral load. The goal of ARV treatment (ART) is to continually keep plasma HIV-RNA levels below the level of detection, 50 RNA viral copies/mL (c/mL), depending on the assay used . Howeverpol). Although reports of transmission of INSTI-resistant virus are rare, as use of INSTIs increases, the potential for transmission of INSTI-resistant virus may also increase. Therefore, when INSTI resistance is suspected, providers should supplement standard baseline genotypic resistance testing with genotypic testing for resistance to this class of drugs [Standard genotypic drug-resistance testing in ARV-naive persons involves testing for mutations in the protease (PRO) and reverse transcriptase (RT) regions in HIV-1 polymerase  which enables the detection of drug resistance mutations (DRMs) present in at least 20–30% of the viral population to be detected , 10. HowRecent Ultra-Deep Sequencing (UDS) or Next-Generation Sequencing (NGS) technologies are known to have, in the majority of cases, better sensitivity and reproducibility than the SS method. NGS technologies perform millions of parallel sequencing reactions to generate many, typically short, reads per run. These technologies can detect and quantify low-frequency variants down to 5% of the total virus population –15.The introduction of NGS into clinical laboratories has been slow for several reasons: (i) the impact on drug response and thus the clinical relevance of low-frequency DRMs before ART and after virological failure remains open to debate , 16–19, Vandenhende M-A et al.  reportedHowever, a recent study by Raymond S et al., using the NGS 454 GS-FLX system, showed that drug-resistant minority variants had no impact on the virological failure of treatment-naive patients to a rilpivirine-based regimen .Few studies have been conducted to compare and evaluate NGS and to determine its detection limit for HIV DRMs, to acquire expertise in performing analyses, or to collect data on which percentage of minority variants is predictive of treatment efficacy.A few years ago, Mohamed and his team has compared HIV-1 DRMs minority variants after virological failure between SS and NGS. They demonstrated that NGS detected all mutations found by SS and identified also additional resistant variants .Recently, Trabaud and his team  performeSentosa SQ HIV genotyping assay  is a novel deep-sequencing, in vitro semi-automated and standardised system. The objective of this study was to evaluate this novel NGS HIV-1 drug resistance monitoring system in a population of HIV-1 infected patients representative of those followed in clinical routine. More precisely, we analysed the repeatability (intra- and inter-run), the precision and the sensitivity of this NGS workflow. We also investigated DRMs and low-prevalence mutations detected by NGS compared to the SS as the reference method with the goal to accreditate this platform in our clinical routine.The A total of 67 plasma samples collected from 40 patients . A nested PCR was performed. The sequences of the amplification primers  were represented in the The sequencing was performed using the big dye terminator v3.1 ready reaction mix on ABI3500 automated sequencer . The PRO, RT and INT sequences were determined using SS according to the Stanford consensus method. Sequences were proofread using the SmartGene HIV module  and the sequence editing was confirmed manually. Mutations compared to HIV-1 Consensus B reference were reported with this module as well as the drug resistance interpretation.The assay was performed according to the manufacturer’s instructions . The Sentosa SQ HIV genotyping assay is reserved for the detection of HIV DRMs in PRO, RT and INT regions from plasma samples. The NGS workflow automates nucleic acid extraction from 730uL plasma samples and PCR set-up (Sentosa SX101), followed by an off-board PCR amplification . The workflow continues with normalisation, enzymatic shearing, purification and adapter ligation (Sentosa SX101). Finally, the two last steps consist of template preparation and sequencing, based on ion torrent technology (Sentosa ST101 and SQ301).The assay contains several controls: a system control (SC), a positive and a negative control. The SC is added as a sample to control the entire workflow from RNA extraction to sequencing data analysis (functions as a positive control), as well as ensuring the absence of contamination in the workflow (functions as a negative control). An internal control (IC) is also added to each sample at the beginning of the extraction. The IC is a positive control for both nucleic acid extraction and library preparation steps. The third control is the Ion Dx CF-1 control, added during the emulsion. This control checks the steps of template preparation and sequencing.+ CEM-derived human T-cell line  as an internal quality control (IQC) (diluted in HIV-negative plasma).A standard panel, HIV-1 drug resistance EQA (QCMD-2016) (n = 5), was purchased form Quality Control for Molecular Diagnostics . These external quality control samples were tested to assess HIV-1 protease and reverse transcriptase sequencing performances. Consensus sequences provided by QCMD were obtained by aligning the sequences of all participating laboratories, mainly European laboratories in the HIV field. A sample from INSTAND-2015 (n = 1)  external quality control was tested to control the integrase region. We also tested an 8E5 HIV positive cell line, derived from LAV-infected cells, a CD4The HIV-1 subtype and drug resistance analysis were determined in 67 clinical specimens and standard panel members by in house SS assay. These results were compared with those generated by Sentosa SQ HIV genotyping assay. Samples were previously genotyped on ABI3500 automated sequencer  and sequences were analysed with IDNS-HIV1 SmartGene module .FASTA sequences were compared using raw data from Sentosa-Vela.DRMs  and drug resistance reports were compared by exporting raw FASTQ sequences from Vela and analysing them with the specific UDS-HIV SmartGene module, IDNS-ASP . Reads were filtered to eliminate low quality reads . An ambiguity filter was used in order to remove uncertain nucleotides. This process is automated and the final alignment can be finally visualised.An interpretation of the genotypic drug resistance report, by using the Stanford 8.4.0 algorithm, was generated with detected mutations and their percentages. The nucleotide ambiguity filter was set at 0.5% and the threshold for interpretation of resistance at 5% when the position was covered by at least 50 reads.The proportions of mutations detected by NGS and SS, categorised according to the percentage of minority variants population, were compared using the ANOVA-test (p<0.05).Intra-assay repeatability was evaluated by analysing patient samples twice in a single run (n = 3) . Inter-aAll real mismatches were at the end of the sequences and as a consequence did not affect the drug resistance reports.All mutations (non-DRMs and DRMs) were correctly identified with SS and NGS methods. The NGS has identified some DRMs, who were not identified by SS method, in only one of the duplicates  was not detected in one of the duplicates. This discordant result (for DRMs and drug resistance reports) is reported in the section 3.6 and in the discussion section.8E5 IQC was used to evaluate the reproducibility. Indeed, one mutation was followed in each region; PRO, RT and INT regions . More prA total score of 340/340 was obtained for the QCMD-2016 (01–05) external quality controls for HIV-1 PRO and RT regions . The QCMThree of five QCMD-2016 samples presented resistance mutations . All DRMMoreover, 8E5 IQC was diluted successively 1/2, 1/5 and 1/10  and compared to SS results. All mutations found in SS were correctly identified with NGS. We obtained 100% of sequences homology, with no mismatch. Moreover, there was no difference in resistance reports between SS and NGS methods (date not shown).The limit of detection (LOD) was estimated with the 8E5 IQC. IQC was diluted to obtain a viral load of 200 and 100 c/mL , with asBased on this IQC analysis, patient samples (n = 10) with a viral load between 285 to 975 c/mL  were selected to establish a LOD. The viral loads 3-fold lower than analytical sensitivity established by Vela Dx (1000 c/mL) were evaluated. All samples were appropriately amplified and sequenced (in term of number of reads and coverage) and the LOD was set at 300 c/mL . The warThis comparison allows the evaluation of the ability of detecting a mutation in one assay compared the other. A total of 614 differences were identified in the 41 samples (40 HIV-1 infected patients and one IQC) , using tThere were 60 differences identified using the SS method compared to the NGS method . These dWe detected 1516 mutations that were identified with both methods (SS and NGS), 357 of which (23.55%) were minority variants (frequency between 5 and 20%).Drug resistance interpretation from the SS and NGS data was compared using the Stanford 8.4.0 algorithm Fig , 2B & 2COne mutation was only identified by NGS at 66.2%, the 138A, conferring resistance to Raltegravir (RAL), Elvitegravir (EVG) and Dolutegravir (DTG). The analysis of electropherograms revealed that this mutation was only present in the forward strand and therefore was not taken into account in the interpretation. The analysis was repeated a second time (undiluted) and the mutation was confirmed.Two mutations were only identified by the SS method. The first DRM was the 263K, conferring resistance to Raltegravir (RAL), Elvitegravir (EVG) and Dolutegravir (DTG). The analysis of SS electropherograms revealed that the background was high. The analysis was repeated (undiluted and diluted 1/10) and in both cases this mutation was not really present. Moreover, the importance of this DRM remains unclear according to the interpretation algorithms. The second DRM was the 103N (conferring resistance to Efavirenz and Nevirapine), the SS analysis showed that this mutation was indeed present. At this time, the possible explanation was the non-detection of a minority variant <20% for a viral load <1000 c/mL.In the 8 discrepant results, 6/8 (75%) were RT mutations, with a 50:50 proportion of NRTI/NNRTI.FASTA sequences using SS and NGS were compared for 40 samples and 6 external quality controls . In both cases, we analysed low-prevalence DRMs that were undetected in one of the duplicates .The criteria of Vela-Dx were not fulfilled for only one sample. Indeed, despite a viral load >1000 c/mL and correct coverage at this position, a mutation presents at 32% (>20%) was not detected in one of the duplicates. The analysis of the Bam files (raw data from the sequencer) by Vela-Dx revealed that this sample had at least three virus subpopulations. In the duplicate in which the mutation could not be detected, only two subpopulations could be amplified. In addition, this specific mutation was not identified with SS method.Vela Dx criteria were fulfilled for other samples evaluated. Indeed, DRMs present at > 20% were detected for viral loads of 1000 c/mL, those >5% and <20% were detected randomly for viral loads below < 4000 c/mL.ARV drug resistance testing is recommended for all HIV-1 infected patients before treatment initiation and after treatment failure , 8. In cSentosa SQ HIV genotyping assay by Vela-Dx is a new, automated NGS-based assay that is user friendly without specialised skills and is standardised. The present study provided an evaluation of this novel NGS HIV-1 drug resistance monitoring system in HIV-1 infected patients and compared it with the SS reference method. This evaluation provided interpretable data for samples regardless of viral loads, virus subtypes or patient history.The The LOD of the NGS sentosa assay was established at 300 c/mL. This limit was three-fold lower compared to the analytical sensitivity established by Vela-Dx (1000 c/mL).Moreover, and in accordance to several publications , 10, we The criteria of Vela-Dx were not fulfilled for one discordant result concerning the intra-run repeatability. Indeed, despite a viral load >1000 c/mL and correct coverage at this position, a mutation presents at 32% (>20%) was not detected in one of the duplicates.The analysis of the raw data from the sequencer revealed that this sample had at least three virus subpopulations. In the duplicate in which the mutation could not be detected, only two subpopulations could be amplified. In addition, this specific mutation was not identified with SS method.The comparison between the two methods (SS and NGS) demonstrated that nearly half (41.86%) of the differences in the mutations identified by NGS were minority variants (occurring at frequencies of 5 to 20%). Only 23.55% of mutations were minority variants that were detected by both methods, highlighting that NGS detects twice as many minority variants, as does SS. Moreover, the majority of these mutations (86.3%) were located in the RT region.A total of 60 mutations were only found with the SS reference method. The detection of most of these (76.87%) can be explained by the fact that the SS method remains a subjective expertise test, especially when the quality of sequences is poor, which may lead to divergent results. A small proportion of these differences (15%) may be explained by the co-existence of variants, generating SS electropherograms that can often be confusing. NGS can distinguish combinations where a codon is really encoded and can discern exact mixtures of more than two amino acids, which is not possible with SS.The comparison of DRMs, by interpretation of drug resistance reports, confirmed that NGS more frequently detected minority variants, 75% of which were present in the RT region. This observation is in accordance with several studies that describe the abundance of low-frequency DRMs detected by NGS in treatment naive patients  as well Among the analysed samples, 80.5% had identical drug resistance reports by NGS and SS methods. Discrepant results were obtained for 8 samples. Five of these samples presented minority variants with an allelic frequency below the limit of detection of the SS method. One mutation was only identified by NGS with an allelic frequency of 66.2%, the RT 138A. The analysis of electropherograms revealed that this mutation was only present in the forward strand and therefore was not taken into account in the interpretation. Two mutations were only identified by the SS. The first DRM was the INT 263K. The analysis of electropherograms revealed that the background was high  and this mutation was not really present. The second DRM was the RT 103N, the SS analysis showed that this mutation was indeed present. At this time, the most probable explanation was a problem linked to this specific sample.At the 5% threshold for reporting minority variants, NGS appeared to attain an increased sensitivity for detecting low-frequency DRMs without compromising sequence accuracy for viral load >1000 c/mL.Moreover, using this NGS assay, we identified novel DRMs that affected the HIV-1 drug resistance interpretation report and may have a clinical impact.This study has some limitations including that the platform was evaluated using a limited number of retrospective HIV-1 patient samples and discordant results were observed for minority variant DRMs and with low viral load.Sentosa SQ HIV Genotyping Assay in a clinical laboratory in Belgium. The NGS appears to be a promising tool for the detection and quantification of DRMs in HIV-1 infected patients. Use of NGS for resistance genotyping can provide useful information in routine clinical practice. Further studies assessing the clinical relevance of low-prevalence DRMs are needed.In conclusion, this is the first clinical evaluation of the


Inflammation has been associated with increased morbidity and mortality in HIV-positive patients. We compared inflammatory biomarkers with dual therapy using lopinavir/ritonavir plus lamivudine (LPV/r+3TC) versus triple therapy using LPV/r plus two nucleoside reverse transcriptase inhibitors (LPV/r+2NRTIs) in treatment-naïve HIV-positive adults.This was a substudy among Argentinian participants in the randomized trial GARDEL. We measured hsCRP, IL-6, MCP-1, TNF, D-dimer and sCD14 from plasma collected at baseline, week 24 and week 48. Generalized estimating equations with an identity/logit link were used to model the average impact of dual versus triple therapy on each biomarker over time, controlling for baseline levels. Additional models estimated the average effect of virologic suppression on biomarker levels over time, adjusting for age, sex, and baseline CD4 count.3. Dual therapy was not associated with significantly different biomarker levels over 48 weeks relative to triple therapy. Virologic suppression was associated with statistically significant decreases in MCP-1, TNF and D-dimer levels and an unexpected increase in sCD14 levels. No change was observed in hsCRP or the proportion of participants with undetectable IL-6 levels.Of 191 trial participants enrolled in Argentina, 172 had baseline and follow-up measurements and were included. Median (IQR) age was 35.5  years and CD4 cell count was 310  cells/mmIn addition to having virologic non-inferiority, LPV/r+3TC dual therapy is generally associated with similar inflammatory biomarker levels over 48 weeks compared to LPV/r+2NRTIs triple therapy in treatment-naïve adults. Further study of dual treatment regimens is warranted. Combination antiretroviral therapy (cART) for HIV infection has traditionally relied on three-drug regimens containing two nucleos(t)ide reverse transcriptase inhibitors (NRTIs) with a third drug from another class., 2 HowevGARDEL  was an open-label randomized controlled trial that demonstrated the virologic non-inferiority of an experimental dual therapy regimen of lopinavir/ritonavir with lamivudine (LPV/r + 3TC), compared to a standard triple therapy regimen of LPV/r with two NRTIs in treatment-naïve HIV-positive adults. At 48 weeks, 88.3% of participants in the dual therapy arm had HIV RNA <50 copies/mL, versus 83.7% of those in the triple therapy arm, giving a difference of 4.6%  and meeting non-inferiority criteria.[Because HIV infection is characterized by increased levels of immune activation and systemic inflammation that are associated with morbidity and mortality, it is also important to consider the relative impact of novel treatment strategies on inflammatory biomarkers. Prior studies have found that inflammation decreases but does not normalize with the use of suppressive antiretroviral therapy,, 8 and tWe conducted a post-hoc sub-analysis of the GARDEL study, an open-label, phase III clinical trial conducted at 19 centres in six countries. Full study details have been published elsewhere. The GARDHIV-1 infected adults were eligible for participation in the GARDEL trial if they were aged >18 years, antiretroviral-naïve with HIV RNA >1000 copies/mL, hepatitis B surface antigen seronegative, and in generally good medical condition. For feasibility reasons, only participants from the nine clinical trial sites in Argentina were eligible for inclusion in this sub-analysis; participants also needed to have plasma specimens available from the baseline visit and at least one of the week 24 and/or 48 visits for analysis. Baseline demographics of the 191 participants enrolled at the nine study sites in Argentina were compared to 227 participants enrolled at the ten sites in other countries  using Wilcoxon rank sum tests for continuous variables and chi-square tests for nominal variables. The baseline demographics of participants from Argentina who were excluded due to lack of follow up were compared to included patients using similar methods.The primary outcome of this analysis was highly sensitive C-reactive protein (hsCRP), an acute phase reactant whose transcription is up-regulated by hepatocytes, smooth muscle cells and macrophages in response to inflammatory cytokines. IncreaseSecondary outcome measures included three inflammatory cytokines: interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and tumour necrosis factor (TNF), which play important roles in leukocyte and endothelial cell activation; as well as D-dimer, a marker of thrombogenesis; and soluble CD14 (sCD14), a marker of monocyte activation. MCP-1 was of interest as it has been associated with hepatic and atherosclerotic comorbidity in people living with HIV., 17 TNF Specimens were stored at -80°C and shipped on dry ice to the laboratory at the University of Toronto, Canada, for batched testing. Plasma concentrations (dilution factor) of hsCRP (1:2000), IL-6 (1:2), MCP-1 (1:2), TNF (1:2), D-dimer (1:5) and sCD14 (1:300) were measured by ELISA  according to the manufacturers’ instructions. Each sample was tested in duplicate, and the results averaged; those with coefficients of variation (CV) exceeding 30% were repeated. Samples with optical densities below the lowest detectable standard were also assigned the value of that standard. Lower limits of detection for each analyte were as follows: hsCRP (0.0139 ng/mL), IL-6 (0.3 pg/ml), MCP-1 (4.0 pg/ml), TNF (0.3 pg/ml), D-dimer (4.6 ng/mL), sCD14 (0.25 ng/mL).Generalized estimating equation (GEE) models using exchangeable correlation matrices and an identity or logit link as appropriate were used to evaluate the effect of dual therapy on each outcome measure. BiomarkeWe also conducted a sensitivity analysis in which participants who did not have HIV RNA<50 copies/mL at week 48 were excluded. Further, to account for a potential effect of concomitant anti-inflammatory medications on biomarker levels, we conducted additional sensitivity analyses in which use of any such drug within 1–4 days of study visits  was included in the model as a time-dependent covariate.Finally, we quantified the effect of having plasma HIV RNA <50 copies/mL on each biomarker at each visit in both treatment groups combined, using GEE models adjusted for age, sex, and baseline CD4 count. These covariates were selected due to prior literature suggesting them to have potential relevance to HIV-associated inflammation.The expected standard deviation (SD) in the primary outcome measure hsCRP was estimated at roughly 4 ug/mL based on published literature. Using eqOf 191 participants randomized for the GARDEL trial in Argentina, 19 had insufficient follow-up specimens for laboratory testing, leaving 172 participants in this analysis. The 191 participants from Argentina were older , were less likely to be male  and MSM , and had higher baseline HIV RNA  than participants from other countries. The 172 participants included in the analysis were similar to the 19 excluded participants in terms of age, gender, risk factor, and baseline CD4 count and HIV RNA.3 and baseline log10 plasma HIV RNA was 5.15  copies/mL. The most commonly selected NRTI backbone in the triple therapy arm was AZT/3TC, at 88%. None of the participants had symptomatic AIDS at baseline. Baseline characteristics of the study population were similar in the dual and triple therapy groups, as summarized in Participants were mostly Hispanic/Latino heterosexual males. Median (interquartile range) age was 35.5  years, baseline CD4 count was 310  cells/mmThe proportion of participants with specimens available for laboratory analysis was 100% at baseline, 97.7% at week 24 and 94.8% at week 48. There was no missing data for baseline values, time, or treatment received. Of the 503 specimens analyzed for each biomarker, 80 (15.9%) were assigned the LLOD value for IL-6 due to having a reading below the LLOD and/or CV>30%; this biomarker was therefore analyzed as a binary outcome in the GEE models. We similarly assigned values equal to the LLOD for seven (1.4%) TNF results and two (0.4%) D-dimer results that were undetectable. None of the hsCRP, MCP-1 or sCD14 results had undetectable results.Median baseline values for each biomarker in the entire sample are shown in Sensitivity analyses excluding the four participants with detectable HIV RNA at week 48 did not qualitatively impact the results. Further, 8.3% and 8.6% of participants had used an anti-inflammatory medication within five drug elimination half-lives of their week 24 and 48 visits, respectively. Sensitivity analyses in which such medication use was considered in the models as a time-dependent covariate gave qualitatively unchanged results.To further explore the relationship between cART and systemic inflammation, we quantified the effect of having plasma HIV RNA <50 copies/mL on each outcome measure . In GEE In this sub-analysis of the GARDEL trial, dual therapy with LPV/r plus 3TC was associated with similar levels of inflammatory biomarkers over 48 weeks compared with LPV/r-based triple therapy in treatment-naïve HIV-positive adults. In analyses that ignored the specific treatment regimen, suppressed viral load was strongly associated with decreased levels of several inflammatory markers, including D-dimer, MCP-1 and TNF. In contrast, HIV RNA <50 copies/mL was associated with an unexpected increase in sCD14 levels, while no association was seen with IL-6 or hsCRP levels.In evaluating the relative merits of novel treatment strategies for HIV infection, it is important to consider a range of different outcomes, including not only virologic response, immunologic recovery, toxicity and tolerability, but also changes in biomarkers of systemic inflammation. That inflammatory biomarkers are similar, rather than higher, in the setting of this dual therapy combination compared to triple therapy is reassuring, because elevated inflammatory markers have been associated with adverse outcomes. For instance, several studies have linked higher levels of hsCRP, IL-6 and D-dimer with higher risks of all-cause mortality,, 22–24 AOur finding of decreases in inflammatory and coagulation biomarkers in the setting of undetectable viremia is generally consistent with prior studies among treatment-naïve patients.–28 Of no3. Further work is needed to ascertain the reproducibility of our finding. Including additional markers of microbial translation such as circulating lipopolysaccharide in future studies could facilitate interpretation of the relationship between undetectable plasma viral load and sCD14; such markers could not be included in this study due to financial constraints.The increase in sCD14 levels with undetectable viremia in our study was unexpected, given prior studies showing a decrease or plateau in this parameter with successful initiation of therapy., 26–29 SPrevious reports from the GARDEL trial have shown that the dual therapy regimen was virologically non-inferior to triple therapy over 96 weeks of follow-up, while also being associated with fewer toxicities and drug discontinuations. While soOther trials have studied dual therapy regimens that combine lamivudine with a potent antiretroviral drug from another class. The ATLAS-M trial randomized 266 patients with plasma HIV RNA <50 copies/mL to either simplify their cART to atazanavir/ritonavir plus 3TC or continue on atazanavir/ritonavir plus two NRTIs. At 48 weeks, 89.5% of dual and 79.7% of triple therapy patients were free of treatment failure, for a difference  of 9.8% , thus meeting pre-specified non-inferiority criteria as well as post-hoc criteria for superiority. This difThree trials have combined lamivudine with the integrase inhibitor dolutegravir. In the exploratory PADDLE study, 18/20 treatment-naïve HIV-positive adults using this regimen had plasma HIV RNA <50 copies/mL at 48 weeks; of the other two, one committed suicide and the other later had HIV RNA <50 copies/mL at the end-of-study visit, without having changed regimens. The GEMIFinally, once-daily oral dolutegravir/rilpivirine and 4-weekly injectable cabotegravir/rilpivirine have both been demonstrated to be viable dual therapy regimens for use as switch strategies virologically suppressed adults, in the SWORD and FLAIR trials, respectively., 45 Key Strengths of our study include the randomized design, variety of biomarkers studied, representing different inflammatory pathways, and large sample size relative to other published studies on inflammatory markers in treatment-naïve patients.Our study also has limitations that warrant consideration. First, our analyses adjusted for a limited number of key covariates  that are felt to impact on systemic inflammation in HIV, and the impact of unmeasured confounders on our results is unknown. For instance, we have previously shown that intercurrent infections and vaccinations can increase hsCRP levels by a clinically important magnitude of 2.244 ug/mL, abnormalIn summary, we found that dual therapy using lopinavir/ritonavir plus lamivudine was associated with similar inflammatory biomarker levels compared with triple therapy using lopinavir/ritonavir plus two nucleoside reverse transcriptase inhibitors in HIV-positive adults over 48 weeks. These findings support the further development of novel dual treatment regimens for HIV.


Cryptococcal meningitis (CM) causes 10%–20% of HIV-related deaths in Africa. Due to limited access to liposomal amphotericin and flucytosine, most African treatment guidelines recommend amphotericin B deoxycholate (AmB-d) plus high-dose fluconazole; outcomes with this treatment regimen in routine care settings have not been well described.Electronic national death registry data and computerized medical records were used to retrospectively collect demographic, laboratory, and 1-year outcome data from all patients with CM between 2012 and 2014 at Botswana’s main referral hospital, when recommended treatment for CM was AmB-d 1 mg/kg/d plus fluconazole 800 mg/d for 14 days. Cumulative survival was estimated at 2 weeks, 10 weeks, and 1 year.3. Two hundred fifteen person-years of follow-up data were captured for the 236 CM patients. Complete outcome data were available for 233 patients (99%) at 2 weeks, 224 patients (95%) at 10 weeks, and 219 patients (93%) at 1 year. Cumulative mortality was 26%  at 2 weeks, 50%  at 10 weeks, and 65%  at 1 year.There were 283 episodes of CM among 236 individuals; 69% (163/236) were male, and the median age was 36 years. All patients were HIV-infected, with a median CD4 count of 39 cells/mmMortality rates following HIV-associated CM treated with AmB-d and fluconazole in a routine health care setting in Botswana were very high. The findings highlight the inadequacies of current antifungal treatments for HIV-associated CM and underscore the difficulties of administering and monitoring intravenous amphotericin B deoxycholate therapy in resource-poor settings. HIV-associated cryptococcal meningitis (CM) causes an estimated 181 100 deaths per year, 73% of which occur in sub-Saharan Africa (SSA) . In contShort- and long-term outcomes with AmB-d under routine care settings in SSA are not well described. Several factors are important in CM management and may affect outcomes even with highly fungicidal AmB-d, including pretreatment hydration, electrolyte supplementation and regular laboratory monitoring for AmB-d-related toxicities , therapeBotswana, a middle-income country in Southern Africa with a 25% adult HIV prevalence and high incidence of CM , 17, hasData were retrospectively collected on consecutive patients admitted to Princess Marina Hospital with laboratory-confirmed CM between January 1, 2012, and December 31, 2014. Princess Marina Hospital is a 530-bed public hospital in Gaborone that serves as 1 of 2 national referral centers for Botswana, providing free treatment to all citizens. The study was conducted in accordance with the ethical standards of the Helsinki Declaration (amended in 2008) and approved by institutional review boards at the University of Pennsylvania, University of Botswana, and Princess Marina Hospital, as well as the Botswana Ministry of Health’s Health Research and Development Committee. A waiver of informed consent was obtained as we collected routine retrospective data and as individual-level patient data were anonymized for the analysis. We included all patients who had laboratory-confirmed CM by either cerebrospinal fluid (CSF) India ink stain, cryptococcal antigen (CrAg) latex agglutination test, or fungal culture, with no exclusion criteria. Patients re-admitted with a recurrence of CM symptoms and laboratory-confirmed CM at any time point after their initial admission were classified as relapse episodes. During the study period, the recommended treatment for CM was amphotericin B deoxycholate 1 mg/kg/d IV plus fluconazole 800 mg/d orally for 14 days, followed by standard fluconazole consolidation and maintenance therapy. Electrolyte supplementation was given at the responsible physician’s dicretion. Antiretroviral therapy (ART) was freely available in the hospital and at public sector clinics, with tenofovir, emtricitabine, and efavirenz as firstline.Cases of CM were identified through review of prospectively collected laboratory records. Demographic information and laboratory results, including blood tests, CSF results, HIV status and associated laboratory results, and basic clinical information including ART use and admission and discharge dates, were then retrospectively obtained for all patients from the electronic medical records system. More detailed clinical history and examination findings, CSF opening pressure recordings, and CM treatment details were obtained from detailed review of paper hospital records, where available, using a standardized data collection form. Time from hospital triage to initiation of antifungal therapy, duration and cumulative dosage of antifungal therapies, number of missed drug doses, frequency of toxicity monitoring, and details of management of raised intracranial pressure were recorded for each CM episode. Mortality data for all patients were collected by linking laboratory records to the electronic national death registry at the Botswana Ministry of Labour and Home Affairs using unique individual identifiers  at the end of December 2015. Cumulative mortality at 2 weeks, 10 weeks, and 1 year after hospital admission with CM were recorded.2 testing. Cumulative survival was estimated at 2 weeks, 10 weeks, and 1 year after initial admission for all patients  and displayed graphically on a Kaplan-Meier curve. Sensitivity analyses were performed assuming all missing patients either survived or died at the point of censoring. Cox proportional hazards models were constructed to explore factors associated with mortality. Unadjusted and adjusted hazard ratios were estimated for predictors of interest, and P values were derived using the likelihood ratio test. Statistical significance was defined as a P value ≤.05. Analyses were performed using Stata, version 13 .Baseline demographic, clinical, and laboratory variables were summarized using descriptive statistics , and proportions were compared using χ3. Seventy-five percent (177/236) of patients had documentation of HIV infection before their initial CM admission, diagnosed a median (IQR) of 14 (2–63) months earlier. Of the known HIV-infected individuals with treatment data available, 57% (81/141) were taking ART at admission, started a median (IQR) of 31 (6–156) weeks before. Baseline laboratory data were available for all patients  and visual disturbances . Comorbidities were common, with a history of treated tuberculosis (TB) in 27% of cases (n = 48) and concurrent TB treatment in 9% (n = 16).During the study period, there were 283 episodes of CM among 236 individuals  . The medpatients . Eighty-All patients were treated with AmB-d (at a dose of approximately 0.7–1 mg/kg/d) plus fluconazole 800 mg/d during the study period. Patient weights were not reliably documented in the medical notes, and the majority of patients (81%) received a dose of 50 mg/d, equating to a single vial of AmB-d. There was a median (IQR) of 2 (1–3) days from presentation to administration of the first AmB-d dose . Over haP < .01) and Grade 3 hypokalemia , but not in patients developing DAIDS Grade 3 or 4 renal impairment.Missed AmB-d doses were more frequent in patients developing DAIDS Grade 4 anemia during treatment  at 2 weeks, 224 patients (95%) at 10 weeks, and 219 patients (93%) at 1 year after initial presentation with CM. Overall mortality was 26%  at 2 weeks, 50%  at 10 weeks, and 65%  at 1 year . In sens9/L), renal impairment (creatinine >110 µmol/L/>1.24 mg/dL), hyponatremia (<125 mmol/L), and low CSF white cell count (<20 cells/µL) (P < .01 for both comparisons) , older age (>50 years), anemia (<7.5 g/dL), raised peripheral white cell count (>10 × 10ells/µL) . There wells/µL)  despite ells/µL) . In multells/µL) . ART staarisons) . Missingarisons) . This asMortality following HIV-associated cryptococcal meningitis in a routine health care setting in Botswana was very high, with only 35% of patients surviving to 1 year. The comprehensive national death registration system in Botswana, combined with an electronic medical records system, provided the unique opportunity to accurately determine long-term outcomes in a large observational cohort of patients treated with amphotericin B deoxycholate therapy given with high-dose fluconazole under “real-world” routine care conditions in sub-Saharan Africa. These poor patient outcomes, in 1 of the better-resourced health care systems in Africa with good access to antiretroviral therapy, highlight the inadequacies of current antifungal treatments for HIV-associated cryptococcal meningitis. The findings also underscore the difficulties faced by busy clinicians in resource-poor settings in administering and monitoring 14 days of intravenous amphotericin B deoxycholate therapy and managing the frequent drug-associated toxicities, with the majority of patients receiving incomplete treatment courses.Mortality rates in this cohort of patients with HIV-associated cryptococcal meningitis were substantially higher than those reported in recent randomized clinical trials (RCTs) from low-resource settings , 21, 22.In this routine care cohort, toxicity monitoring was performed less frequently than recommended in World Health Organization guidelines , with a Importantly, short-term outcomes observed with amphotericin B deoxycholate plus fluconazole 800 mg/d treatment under usual care conditions were only marginally better than the survival described in prospective cohorts and RCTs from sub-Saharan Africa with high-dose (800–1200 mg/d) fluconazole monotherapy , likely The baseline predictors of mortality in this routine care cohort—older age, abnormal mental status, raised peripheral white blood cell count, hyponatremia, and low CSF white blood cell count—have previously been described in prospective cohort studies . SeventyGiven the high ongoing burden of HIV-associated cryptococcal meningitis in Africa , 16 and Another promising strategy to reduce mortality, currently being evaluated in a phase III study (ISRCTN 72509687), is administration of single high dose of liposomal amphotericin B. Phase II data have shown single 10-mg/kg doses to be safe, with similar early fungal clearance compared with 2 weeks of standard liposomal amphotericin B dosing . In paraOur study has several important limitations, primarily relating to the retrospective data collection. Botswana’s electronic death registry and electronic laboratory records enabled near complete ascertainment of 1-year vital status data and laboratory results. However, we were still dependent on retrospective medical record review to collate several key clinical variables. Paper medical records were missing in a substantial proportion of patients, limiting clinical data availability. Patient weights were rarely available, precluding any assessment of potential weight-based underdosing or overdosing of amphotericin. The median weight in the cryptococcal meningitis patients in our clinical trials in these settings (IQR) was 52 (45–61) Kg , with a In conclusion, through analysis of registry-based data and electronic medical records, this study has demonstrated the extremely high mortality resulting from HIV-associated cryptococcal meningitis in patients treated with standard amphotericin B deoxycholate and high-dose fluconazole therapy in Africa. Much of this mortality occurs in the first months after hospital discharge and would be missed in hospital-based studies. These findings highlight the urgent need for effective implementation of cryptococcal prevention strategies such as cryptococcal antigen screening, the need for new, easy-to-administer, and effective treatment regimens for cryptococcal meningitis, and the importance of advocacy efforts to ensure that drugs such as flucytosine and liposomal amphotericin B, which will form the key components of these new treatment regimens, are made accessible and affordable in Africa.Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary TablesClick here for additional data file.


South Africa has a high HIV burden. Despite increased uptake of persons living with HIV into the South African national antiretroviral therapy programme, the incidence of HIV increased between 2013 and 2016. Studies suggest that increased community viral suppression results in reduced HIV incidence in that community ‘independent of unsafe sexual behaviours and sharing used syringes’.The aim of this study was to investigate the viral and immunologic responses of patients, in a rural community health centre in South Africa, to combination antiretroviral therapy (cART) between January 2004 and July 2016.p-value < 0.05 was considered to be statistically significant.This was a retrospective medical record review conducted in Thohoyandou Community Health Centre. Data analysis was done using SPSS 24.0 and Microsoft Excel. The estimates used were 95% confidence intervals, and a Analysis was done using 1247 individuals, with 76% of the cohort being female and 98% first-line cART. The proportion of patients with a suppressed viral load (VL) at 6 months post-treatment was 64%, and 72% at 60 months. Fifty-nine per cent had consistent viral suppression over a 6-month period and 14% over at least 54 months. The mean CD4+ cell count at baseline was 227 cells/µL, and 538 cells/µL at 60 months. Multivariate regression analysis revealed that males had poorer immunologic and virologic responses.Viral suppression in the study population was inferior to the UNAIDS target of 90%. The sustainability of viral suppression, once attained, was also low. These may have a negative impact on HIV transmission. Studies have suggested that increased community viral suppression results in reduced HIV incidence in that community ‘independent of unsafe sexual behaviours and sharing used syringes’.5 The Partner study found ‘zero transmission’ when the viral load (VL) at most 1 year before was less than 200 copies/mL.6 To curb its high HIV burden, South Africa adopted the UNAIDS ‘90-90-90 targets’. This target aims to attain sustained viral suppression in 90% of all persons receiving antiretroviral therapy by the year 2020.The prevalence of HIV in South Africa increased from 6.19 to 7.03 million between 2013 and the end of 2015. Despite an increased uptake of persons living with HIV into the national antiretroviral therapy programme over the years, the incidence rate also increased from 340 000 new cases per annum to 380 000 cases per annum during the same period.7 The South African National Department of Health (NDoH), however, recommends the use of VL ≤ 50 copies/mL as definition for viral suppression and two VL results > 1000 copies/mL to define viral failure.8 Bartlett et al. state that maintaining the VL at ≤ 50 copies/mL is associated with the most durable clinical benefits.9 The CD4+ cell response to combination antiretroviral therapy (cART) is, however, more variable. It increases rapidly in the first month of ART by 75 cells/µL–100 cells/µL, with a more gradual response thereafter.10 The CD4+ cell count of some patients, however, fails to increase despite viral suppression. Several causes have been suggested for this ‘immuno-virologic discordant’ CD4+ cell response. Insufficient thymic activity and ongoing viral replication, despite good viral suppression, may account for this.11 Some studies have investigated the implication of immuno-virologic discordance. Zoufaly et al. found that compared with those whose CD4+ cell count increases with viral suppression, patients with immuno-virologic discordance had increased risk for developing AIDS and other complications, especially in the first 6 months of therapy. Several factors have been shown to be associated with viral suppression, adequate CD4+ cell response and immuno-virologic discordance.12Over the years, several definitions have been used to define viral suppression and virologic failure, with different definitions recommended for low- to medium-income countries, compared to high-income countries.13 Other factors have also been shown to be associated with a delay in or failure to attain viral suppression. Devey et al.14 stated that the age group less than 15 years, male gender, prior ART exposure and being on tuberculosis treatment were associated with an increased risk of virologic failure in South Africa. However, in Swaziland, Jobanputra et al.15 found that age group less than 20 years and CD4 count less than 350 cells/µL were associated with virologic failure. In contrast, gender was not an associated factor. Another factor associated with higher incidence of viral failure is a high VL at initiation. A high CD4+ cell count and better clinical stage at initiation are, however, associated with earlier viral suppression.13 Finally, Bello et al.16 found that a longer duration post-cART initiation, less previous antiretroviral drug use before lifelong cART, higher baseline CD4 cell count and lower baseline VL were associated with 10 years of sustained viral suppression.It is known that poor adherence to antiretrovirals is associated with failure to suppress HIV. The best indicator of adherence and response to treatment is virologic response, and more than 90% – 95% adherence is required to achieve viral suppression.8The main objective of this study was to investigate the virologic and immunologic responses of patients, in a rural community health centre in South Africa, to cART, as well as the factors that are associated with these responses. Individuals who had been on cART for less than 6 months were excluded from the study, as the minimum time required to determine response was 6 months.A retrospective review was performed on the database (electronic and paper records) of Thohoyandou Community Health Centre (TCHC), which has one of the highest number of patients on cART in Vhembe District. TCHC is located in Thulamela Municipality in Vhembe District, which is the northernmost district in Limpopo Province, South Africa. It is a public health facility. A data collection form was designed to retrieve the data, including age, sex, marital status, year of initiation, baseline body mass index (BMI), HIV clinical stage, haemoglobin and estimated glomerular filtration rate (eGFR), along with the serial CD4+ cell counts and VL measurements. The study population included individuals initiated on cART between 01 January 2004 and 31 July 2016 and stored on tier.net. This included those transferred into the facility and those initiated at the facility. The database (tier.net) also had some missing data. Many patients did not have any VL and/or CD4 cell count results recorded even after 6 months, and as a result they were excluded from the study. Also excluded were those who defaulted and reinitiated later. Therefore, their duration on cART could not be correctly determined.18Typical first-line antiretroviral regimen consisted of stavudine, lamivudine and nevirapine or efavirenz, prior to 2010. Stavudine was replaced with tenofovir after 2010, with a fixed-dose combination (FDC) introduced in 2012. Second-line highly active antiretroviral therapy (HAART) consisted of abacavir or zidovudine, combined with lamivudine and ritonavir-boosted lopinavir. Third-line regimen was to be decided by a review committee.19 If an individual had more than one VL or CD4+ cell count measurement at a fixed interval, the result closest to the particular interval was used. The sustainability of viral suppression was also reviewed. This was defined as having at least two consecutive VL results that were at most 50 copies/mL. The association of other covariates with these responses was also evaluated.Virologic and immunologic responses were measured up to 132 months after cART initiation. Virologic response was measured using plasma HIV RNA concentrations. A VL of less than 50 copies/mL was considered suppressed. Because of differences in types of VL assays used over the period under review, analysis was also done using a VL less than 400 copies/mL as viral suppression. Immunologic response was measured by CD4+ cell count. An increase in CD4+ cell count of at least 50 cells/µL at 6 months after cART initiation was considered as adequate.Mortality was determined exclusively as recorded on tier.net. However, the recording of death on tier.net was not corroborated with national mortality database. This may have affected the validity of a more detailed analysis.U test was used to check for equality of the median of the continuous variables (duration on cART and CD4+ cell count) as they were not normally distributed. Pearson’s chi-square test was used for categorical variables.The data used for the study were extracted from tier.net. Data not found on tier.net were retrieved from paper folders of individual patients. It was converted, using Microsoft Excel 2010, into a format for analysis in SPSS software version 24.0. Descriptive and inferential statistics were generated. During data cleaning, duplicate entries were identified and removed. The data set for socio-demographic variables and clinical variables were summarised. Continuous variables were presented as means. Categorical variables were presented as percentages or proportions. Mann–Whitney p-values < 0.2 were introduced into the multivariate models, and their adjusted odd ratios derived. The estimates used were 95% confidence interval (CI), and a p-value < 0.05 was considered to be statistically significant.To model for factors associated with virologic and immunologic responses, univariate and multivariate logistic regression analysis was performed. Univariate analysis was performed first, and crude odds ratios for the association of the variables with immunologic and virologic responses determined. The variables included age, year of initiation, gender, marital status, baseline BMI, haemoglobin, clinical stage and estimated GFR. Variables with Face validity was ensured by conducting a review of a sample of the data collection form by non-experts in the field of HIV. Content validity was ensured by carrying out a detailed literature review and reviewing the instrument with an expert in the field of HIV research. Corrections were made based on the reviews.20The reliability of the data collection form was measured using interrater reliability method of reliability testing. The calculated Cohen κ coefficient was 0.79, which was within the acceptable limit.Ethical clearance was obtained from the University of Venda Research Ethics Committee (Protocol number SHS/16/PH/16/1808). Approval was obtained from the Limpopo Provincial Department of Health to acquire and use the data for the study. The data stored on the computer were protected from theft using the most recent spyware protection.The clinical data of 1912 individuals were reviewed. Two hundred and seventy-nine individuals were excluded because they had been on treatment for less than 6 months. Of these, 68 had died. Those excluded for having no results even after 6 months were 177. Then, 209 individuals were not included because they defaulted treatment and were later reinitiated on cART. As a result, the determination of their duration on cART was affected. The clinical data of the remaining 1247 individuals were used for the analysis. Of these, 191 individuals initiated treatment in 2013. This corresponded to 15.4% of the total sample. One hundred and eighty-four individuals (14.8%) were initiated in 2014 and 183 (14.7%) in 2015. Only 0.3% of the cohort were initiated in 2004.p = 0.01). There was no gender difference for those who were LTFU or who defaulted. Throughout the study, at least 98% of the cohort were on a first-line regimen. There was no one on a third-line regimen throughout the period under review.Twenty-four per cent of the cohort were male and the mean age was 36 ± 5 years. Other baseline characteristics of participants are shown in n = 648) were virally suppressed. At 12 months, 70% (n = 577) were suppressed, 70% (n = 453) at 24 months and 69% (n = 392) at 36 months. The proportion virally suppressed almost consistently increased with duration on cART. At 132 months, 94% (n = 16) were suppressed. When the definition of viral suppression was changed to ≤ 400 copies/mL, at least 80% viral suppression was attained at every interval throughout the study (N = 882). Only 14% had consistent viral suppression over a 54-month period. Twenty-eight (2.3%) had two or more VLs >1000 copies/mL at least 6 months apart and still remained on the failing regimen.After 6 months of cART, 64% (he study . Those wp = 0.952) in achieving CD4+ cell count of ≥ 500 cells/µL at 60 months, when individuals with a baseline CD4+ cell count of ≤ 200 cells/µL were compared with those with a baseline CD4+ cell count > 200 cells/µL. The calculated immuno-virologic discordance for this cohort was 27% (n = 415). Also, of those that achieved viral suppression at 60 months, 56.4% achieved a CD4+ cell count.The mean CD4+ cell counts at specific times after initiation on cART from baseline were calculated. The mean CD4+ cell count at baseline was 227 cells/µL. This increased to 372 cells/µL at 6 months. The increases were consistent until at 60 months after initiation, at which point it was 538 cells/µL. At 120 months, the mean CD4+ cell count was 487 cells/µL . Fifty-fp = 0.046) of attaining at least one consecutive viral suppression, as compared to those less than 15 years. In addition, males had 38% lower odds  of attaining viral suppression, as compared to females.p = 0.01) of attaining adequate immunological response, as compared to those less than 15 years.The factors associated with adequate CD4+ cell count response, as shown in 21This study reviewed virologic and immunologic responses of patients, in a rural community health centre in Vhembe District, South Africa, to cART over a 132-month period. It found that most (78.4%) patients were initiated on cART after 2010. This is probably because of changes in the South African NDoH guidelines since 2010, and the fact that patients were being initiated in any clinic closer to their homes. This increased uptake into the antiretroviral therapy programme.22 found that women seemed to have more access to HAART than men and children. This is because pregnancy and breastfeeding are used as entry points for women by testing for HIV and initiation on cART. Presently, no such entry point exists for men and most children.Furthermore, more females were initiated on HAART than males and most people in the cohort were aged 16–44 years. Johnson et al.23 However, data for previous exposure to PEP were obtained by recall of patients before being stored into the electronic database. Also, during transfer into the health facility, information on PEP was missing for some patients. This may account for the low prevalence of PEP exposure.Our study also showed that only 0.9% of the cohort had any form of previous antiretroviral exposure. This proportion is quite low, considering that the guidelines recommending PEP have been available for more than 6 years. This may contribute to the high incidence of HIV in South Africa as, although PEP is not 100% effective, it has been shown to reduce HIV transmission in non-occupational exposures.The results of this study showed a pattern in which the proportion of virally suppressed individuals increased with the passage of time. Also, the data recorded for individuals in this study also decreased consistently with time. This may have created a ‘retention bias’, which ensured that those who were likely to have unsuppressed VLs  did not have VL data used for these measurements. The proportion of individuals virologically suppressed was therefore calculated based on the number of individuals who had data recorded at a particular time point.n = 648), 70% (n = 577) and 72% (n = 366) of our cohort were virologically suppressed at 6, 12 and 60 months, respectively. When the definition of viral suppression was changed to < 400 copies/mL, the minimum proportion of virologically suppressed individuals at every time point throughout the study was 80%. This was similar to a large meta-analysis conducted by Boender and colleagues in low- to medium-income countries. They found that at every interval between 6 and 60 months, at least 80% of patients were virally suppressed.24 This was an unexpected but important finding in the drive towards achieving one of the ‘90-90-90 targets’ set by the UNAIDS. Present cART guidelines recommend initiation once diagnosed with HIV. One of the major aims of this is to prevent HIV transmission through achieving viral suppression, as revealed by the Partner study.6 A review was, therefore, attempted to assess how consistently patients were able to maintain viral suppression after attainment. Fifty-nine per cent of the sample were able to maintain one consecutive viral suppression. These patients maintained viral suppression over a period of at least 6 months. When reviewed for those able to maintain viral suppression consecutively for at least 54 months, only 14% of the sample achieved this. This low proportion of people with consistent viral suppression may have a negative impact on the use of viral suppression as a prevention strategy.Our study found that 64%  at 60 months, unlike in our study.25 in a study to ascertain patient retention on ART after 5 years of treatment in Tshwane District, however, found that gender and age had no influence on early VL response (6 months). Some other studies also found that higher baseline CD4+ cell count was associated with viral suppression, and males, age group less than 15 years and prior ART exposure were more likely to experience virologic failure.14 When the factors that are associated with viral suppression in the long term were reviewed, gender and baseline laboratory results had no significant association. It seemed as if males that were able to stay on treatment for longer had similar virologic response to females. This may have been because of the retention bias, noted above, as more males died or were LTFU during the study. Bello et al.16 also found that absence of prior exposure to antiretrovirals before initiation on lifelong HAART was associated with viral suppression at 10 years post-initiation. They also found that age, race and baseline CD4+ cell count had no influence on viral suppression in the longer term.The only factors found to be significantly associated with early viral suppression were baseline CD4 count and gender, with individuals with higher CD4+ cell counts and females more likely to achieve viral suppression at 6 months. The age group less than 15 years also had comparatively poorer immunological and virologic outcomes. Problems with adherence and drug formulations may account for this. Mlangeni and Senkubuge,Although attempts were made to prevent bias, missing data on tier.net and the use of patient recall to record some variables, such as previous antiretroviral therapy exposure, may have introduced some bias to the study. Furthermore, patients recorded as LTFU may have died. However, because the data were not updated with the South African national mortality database, the cumulative mortality may have been underestimated. Furthermore, being a retrospective study, data on some variables that may influence responses to cART, such as adherence, were not available. This introduces the possibility of residual confounding into the study.Despite the limitations outlined above, this study has a number of strengths. The use of all patients on cART recorded on tier.net affords an opportunity to find a difference in outcomes if there really is one. Finally, there is a paucity of studies reviewing immunologic and virologic responses in the long term in healthcare facilities in Limpopo, South Africa; therefore, this study will serve as a foundational study for such research.26According to the South African NDoH definition for viral suppression, the proportion of individuals attaining viral suppression was well below the UNAIDS 90% target. Also, the sustainability of viral suppression seemed quite low when individuals were reviewed over a 54-month period. Immunologic response was adequate for most individuals in the period under review. Being male was associated with poorer virologic response to cART. Furthermore, the age group less than 15 years had poorer immunologic and virologic outcomes. Therefore, a targeted approach focusing on improving virologic and immunologic responses of males and patients less than 15 years may aid in improving outcomes at a community level because infected male partners are likely to infect a female partner and vice versa.


The objective was to analyze the effectiveness and safety of dual therapy with rilpivirine plus boosted-darunavir (RPV + bDRV) in real-life patients.Observational, retrospective, multi-center study in HIV+ patients who had received RPV + bDRV for 24 weeks to optimize/simplify their previous antiretroviral treatment. We determined the percentage of patients without virologic failure  at 24 weeks of treatment.The study included 161 patients from 15 hospitals with median age of 49 years; 29.3% had previous AIDS stage and median CD4+ lymphocyte nadir of 170 cells/uL. They had been diagnosed with HIV for a median of 17 years and had received 14 years of ART, with five previous treatment combinations, and 36.6% had a history of virological failure. The reasons for the switch were simplification/optimization (49.7%), toxicity/intolerance (17.4%), or inadequate effectiveness of previous ART (10.6%).intention-to-treat” analysis at 24 weeks, 87.6% of 161 patients continued the study treatment without virologic failure criteria.Baseline VL of 50–1000 copies/mL was recorded in 25.5% of the patients. In the“on treatment” analysis  the efficacy was 94.6% (141/149 patients).In the “Dual therapy with RPV + DRVb proved to be effective and safe in patients with advanced HIV infection, long exposure to ART, low CD4 nadir, previous virologic failure, and/or history of ineffective ART. The life expectancy of HIV-infected individuals is approaching that of the general population due to the effectiveness and good tolerability of new antiretroviral drugs.The main concerns over antiretroviral therapy (ART) are no longer efficacy and tolerance, but antiretroviral drug-related toxicity , especiaReverse Transcriptase Inhibitors (NRTI) –8, whoseFor this reason, nuke-sparing regimens (NSRs), that do not include one or both NRTIs, have been developed to reduce and prevent ART-related toxicity . The devNumerous studies  have found that the efficacy of PI monotherapy (MT) –27 and dFewer data are available on the application of DT in patients with a less favorable profile , although they have generally proven high efficacy and a reduction of possible toxicity –62.DT with rilpivirine and boosted darunavir (RPV + bDRV) is an attractive NSR that appears to combine both a high efficacy and genetic barrier with a lower pill burden, good tolerance and toxicity profile. However, despite its utilization in clinical practice, there has been little research on the outcomes.With this background, we performed a retrospective multi-center investigation to analyze the profile of patients prescribed with this combination and study its efficacy and safety. Confirmation that these NSRs are safe and effective will help to consolidate them as an optimized alternative for ART that could improve the long-term prognosis of HIV-infected patients and reduce treatment costs.An observational, retrospective, multi-center study was conducted in HIV-infected patients who switched to DT with rilpivirine (RPV)  and boosted darunavir (bDRV)  (with either ritonavir or cobicistat).The main objective of the study was to analyze the effectiveness of the DT by calculating the proportion of patients with virologic success .Secondary objectives were to establish: a) proportion of patients with RNA HIV-1 < 50 copies/mL at week 24 of treatment; b) stratification of all viral load determinations obtained during exposure to the DT; c) Incidence of new adverse events; d) Impact of the DT on lymphocyte subpopulations, lipid profile, and liver and kidney function; e) Analysis of possible differences in efficacy and potential toxicity between darunavir/ritonavir (DRV/r) and darunvir/cobicistat (DRV/c).Patients from 15 Spanish hospitals were evaluated, with the following inclusion criteria: infection with HIV-1, age > 18 years, and receipt of antiretroviral treatment with RPV + bDRV before December 31st 2015 with a minimum follow-up period of 24 weeks. They were also required to have a baseline viremia at switch (bVL) < 1000 copies/mL and to have signed informed consent to retrieve data from their medical records.A standardized electronic database was use to collect the following variables: age, gender, date of HIV diagnosis, VL at diagnosis, CD4 lymphocyte cell count at diagnosis, CD4 nadir, HIV stage (CDC), date of first ART, number of previous ART combinations, previous ART, previous VF (and ART received and genotype mutations at time of VF), reason for switch to study combination , booster (ritonavir or cobicistat), weeks of exposure to ritonavir and cobicistat, and HIV- 1 RNA, CD4 cell count, and bloodwork at baseline, 4–8 weeks, 9–23 weeks, and ≥ 24 weeks.New adverse events during exposure to DT and reasons for not completing 24 weeks of follow-up were also collected .For the first and secondary objectives of virologic efficacy, an intention-to-treat (ITT) analysis was carried out (considering losses as failures) as well as an on-treatment (OT) analysis, excluding patients who discontinued treatment with the DT for any reason other than VF .Means with standard deviation, medians, and interquartile ranges were calculated for quantitative variables and absolute and relative frequencies for qualitative variables. After applying the Kolgomorov-Smirnov test to check the distribution of variables, effects of the DT on change in analytical parameters  were analyzed using the paired samples t-test when the distribution was normal or the signed-rank Wilcoxon test when it was not. For bivariate analyses, the Student’s t-test was applied when the distribution was normal and the Mann-Whitney test when it was not. SPSS v20.0  was used for data analyses, and the significance level was 0.05 for all tests.The study was approved by the regional ethics committee of Andalusia (CCEIBA) on April 25, 2016 and participants provided written informed consent for data from their medical records to be collected.One hundred and eighty-nine patients switched to RPV + bDRV: 28 did not meet inclusion criteria (20 with baseline VL > 1000 copies/mL and 8 with follow-up of < 24 weeks). Therefore, the final study sample comprised 161 patients with median age of 49 years (IQR 44–53), median time since HIV diagnosis of 17 years (IQR 10–23), receipt of ART for median of 14 years (IQR 6–18), and median of 5 previous ART combinations (IQR 3–7); 29.3% had a history of AIDS (stage C or 3 according to CDC), and the median CD4 lymphocyte nadir was 170 cells/μL (IQR 73–290) [Table At baseline, 25.5% did not have suppressed HIV viremia , although the baseline CD4 lymphocyte cell count was 618 cells/μL (IQR 370–861) [Table The main reason for the switch to a DT with RPV + bDRV was simplification/optimization of their ART (49.7%), followed by previous ART toxicity (17.4%), and insufficient effectiveness of previous ART (10.6%).Initially, the DT boosted with ritonavir (DRV/r) was administered to 131 patients (81.4%), 29 of whom switched to cobicistat (DRV/c) during the first 24 weeks of treatment. A total of 59 patients (36.6%) were exposed to DRV/c during the study period.Figure Excluding the 12 patients who dropped out of the study for reasons other than VF , 94.6% (141/149) completed the study without showing VF criteria.ITT analysis). In 89.3% (133/149) of the OT population, the VL was < 50 copies/mL at 24 weeks .In 82.6% (133/161) of the study population, the VL was < 50 copies/mL at 24 weeks , 50–1000 copies/mL in 11.1%, and > 1000 copies/mL in 1.2%; 237 of these determinations were obtained during exposure to RPV + DRV/r, with 87.3% being < 50 copies/mL, and 97 during exposure to RPV + DRV/c, with 88.7% being < 50 copies/mL.The last observed VL determination of the 159 patients with virologic data after the switch was < 50 copies/mL in 86.2% of patients (< 20 copies/mL in 80.5%), between 50 and 1000 copies/mL in 12.6%, and > 1000 copies/mL in 1.3%.p = 0.071) and the CD4/CD8 ratio by 0.04 from 0.75 to 0.79 (p = 0.004).During the 24 weeks of exposure to the DT, the CD4 lymphocyte cell count increased by 34 cells/mm3 from 630 cells/mL to 667 cells/mm3 .Patients with previous failure while receiving darunavir were not more likely to present VF with the study DT [13.3% (4/30) vs 5.5% (1/18), p = 0.470). There were also no differences in VF rate between those who received the DT boosted with ritonavir vs cobicistat .We analyzed the influence of CD4 nadir on VF and found no statistically significant differences between those who failed and those who didn’t , total cholesterol , HDL-cholesterol , LDL-cholesterol , GOT , GPT , GGT , and alkaline phosphatase  but not in total cholesterol/HDLcholesterol ratio (Table p = 0.462).Statistically significant differences between values at baseline and 24 weeks were found in creatinine  and/or high viral loads and few drug resistance tests were performed.Despite the disadvantageous profile of our study population, the proportion of virologic suppression obtained with this DT was similar to that obtained with TT (including stable patients with no history of VF and with suppressed viremia in this switch scenario). Stratification of all viral load determinations in the entire cohort during the study period showed similar rates of blips and VFs, and almost all of the latter could be attributed to poor treatment adherenceThis is a preliminary analysis of a cohort that we are still following, but we believe that a ‘24 weeks Analysis’, considering a threshold of 50 copies/mL for VF, is enough to determine virologic effectiveness for previously suppressed patients. This is the minimum timeframe required to prove ART’s ability to suppress viral replication in naïve patients –68 and fDespite their long history of ART, the immunological recovery was similar to that reported for TT, with an increase in CD4 lymphocytes and CD4/CD8 ratio.Tolerance of the combination was generally good, although several patients asked to switch to previous or alternative treatments due to toxicity, which was considered mild in all of these cases.A slight increase in total cholesterol and LDL-cholesterol levels was observed with the DT under study; however, there was also an increase in HDL-cholesterol levels, with no change in the atherogenic index over the 24-week observation period. There was a significant decrease in transaminase levels, implying a reduction in the potential toxicity of this DT, which supports the idea that NRTIs such as tenofovir could have certain level of hepatotoxicity.More than one-third of the patients received DRV/c  and showed no difference in safety and effectiveness outcomes with those receiving DRV/r.Study limitations include its retrospective, multi-center design, although the necessary data were recovered for almost all patients. Inclusion bias was minimized by recruiting all patients who had been prescribed with the DT under study in the participating hospitals. However, it was not possible to evaluate potential long-term changes in the toxicity of this combination due to the study design and short follow-up period.Dual therapy with RPV + bDRV in the clinical setting has proven to be effective, even in patients with advanced HIV infection, extended exposure to ART, low CD4 lymphocyte nadir, history of VF, and/or previous non-suppressive ART.