In the Western world, approximately 30% of HIV-infected individuals are still very late presenters. The aim of this retrospective study is to describe epidemiological and clinical characteristics, as well as long-term immunological outcome in virologically suppressed HIV+ very late presenters.We reviewed the medical records of all consecutive HIV+ patients with CD4 cell count < 200/ mmc3 at presentation, who had attended our clinic between 1996-2006, and had achieved a persistent virological suppression for at least 1 year. Demographic, clinical, virological and immunological data at baseline and at follow up visits were collected. The changes in CD4+ cell count during follow up was also examined, stratifying the population according to baseline age, HIV risk factors, CD4+ cell counts, HIV viral load , HCV co-infection.Overall 164 very late presenters with a persistent virological response were examined. Caucasian and heterosexual males represent the largest part of this cohort of virological responder, very late presenter patients. IDUs and HCV coinfected patients were under-represented as compared to our HIV population, probably due to a lower adherence. Epidemiological and clinical characteristics of the study population: 61.2% had CD4+ cell count <50/µl. 123 patients started a protease inhibitor-based regimen (75%). Respectively 25% and 52% of initial NNRTI and PI-based regimen were modified during follow up (toxicity was the most common cause of switch). After 5 years of therapy a good immunological recovery (> 500 CD4 cell/µl) was observed in 30.7% and 46% of patients with baseline CD4+ cell count < 50/µl and 51-200/µl respectively.CD4+ cell count increased even after 5 years, reaching a full immunological recovery (>700/µl) only in 17% of patients. Patients aged ≥ 50 years, IDUs and HCV co-infected had a slower and/or lower immune recovery; no significant differences in immunological response according to baseline viral load were observed.A fair immune recovery over 5 years of HAART was seen. The CD4+ cell count restoration was conditioned by baseline values, age, HCV coinfection, and a complete immunological recovery was achieved in a very limited subset of patients.


Clinical relevance of low-frequency HIV-1 variants carrying drug resistance associated mutations (DRMs) is still unclear. We aimed to study the prevalence of low-frequency DRMs, detected by Ultra-Deep Sequencing (UDS) before antiretroviral therapy (ART) and at virological failure (VF), in HIV-1 infected patients experiencing VF on first-line ART.Twenty-nine ART-naive patients followed up in the ANRS-CO3 Aquitaine Cohort, having initiated ART between 2000 and 2009 and experiencing VF  >500 copies/ml or one VL >1000 copies/ml) were included. Reverse transcriptase and protease DRMs were identified using Sanger sequencing (SS) and UDS at baseline (before ART initiation) and VF.Additional low-frequency variants with PI-, NNRTI- and NRTI-DRMs were found by UDS at baseline and VF, significantly increasing the number of detected DRMs by 1.35 fold (p<0.0001) compared to SS. These low-frequency DRMs modified ARV susceptibility predictions to the prescribed treatment for 1 patient at baseline, in whom low-frequency DRM was found at high frequency at VF, and 6 patients at VF. DRMs found at VF were rarely detected as low-frequency DRMs prior to treatment. The rare low-frequency NNRTI- and NRTI-DRMs detected at baseline that correlated with the prescribed treatment were most often found at high-frequency at VF.Low frequency DRMs detected before ART initiation and at VF in patients experiencing VF on first-line ART can increase the overall burden of resistance to PI, NRTI and NNRTI. The advent of combination antiretroviral therapy (ART) has dramatically reduced HIV-1 infection-related morbidity and mortality Standard genotyping by Sanger sequencing (SS) used currently in clinical practice cannot detect viral variants representing less than 15–25% of the viral population Clinical relevance of detecting low-frequency DRMs remains open to debate. Some studies have found no significant association between the presence of low-frequency DRMs and subsequent virological failure The objectives of our study were to determine the prevalence of DRMs detected by UDS as well as their effect on ART resistance before treatment and at VF, and to analyse their evolution under treatment, in HIV-1 infected patients experiencing VF on first-line ART.All patients included in this study gave written informed consent. The study protocol was approved by the Ethics committee of Bordeaux University Hospital (Comité de protection des personnes).The Agence Nationale de Recherche sur le SIDA (ANRS) CO3 Aquitaine Cohort is a prospective hospital-based cohort of HIV-1 infected patients under routine clinical management, initiated in 1987 in the Bordeaux University Hospital and four other public hospitals in the Aquitaine region, South Western France. Inclusion criteria are: adult patients of the participating hospital wards with confirmed HIV-1 infection, having at least one follow-up after the first report, and having given informed consent. Visits occur usually every three months if the patient is treated, every six months otherwise. Detailed presentation of the cohort has been reported elsewhere Patients starting a first antiretroviral treatment between January 2000 and June 2009 were retrospectively screened from the ANRS CO3 Aquitaine Cohort database.Patients experiencing virological failure (VF), defined as a plasma viral load (VL) >1,000 copies/ml or 2 consecutives VL>500 cp/ml at least 6 months after ART initiation, and with plasma samples available both at baseline (last sample available before ART initiation) and at VF (plasma sample corresponding to first VL>1000 or second VL>500 cp/ml) were included in our study. Patients were excluded if they changed or stopped their ART. Socio-demographic, clinical and biological characteristics of eligible participants were extracted from the database.http://www.hivfrenchresistance.org).Viral RNA was extracted from 1 ml of plasma sample at baseline and at VF using the High Pure Viral RNA kit (Roche). The Reverse Transcriptase (RT) and Protease gene sequences were determined using Sanger sequencing (SS) according to the ANRS consensus method (Pol region (HxB2 position: 2082–3420) was amplified using the 5P1 and MJ4 primers and the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase kit . RT-PCR conditions were reverse-transcription at 50°C for 30 minutes, one denaturation step of 94°C for 2 minutes, followed by 40 cycles of denaturation for 30 seconds at 94°C, annealing for 30 seconds at 55°C, extension for 90 seconds at 68°C, and a final 7-minutes extension at 68°C.Viral RNA was extracted as described above and used as a template for RT-PCR. The Two microliters of RT-PCR product were used to generate 4 amplicons  encompassing protease and RT regions and harbouring specific MIDs in their end. Amplicons and primers are described in PCR products were purified using magnetic beads  to eliminate primer-dimers. The number of molecules was quantified by fluorometry  and equimolar concentrations of amplicons were pooled. This library was submitted to emulsion-based clonal amplification (emPCR) using GS Junior Titanium emPCR Kit (Lib-A) according to Roche's protocol. UDS was performed in both strands on a 454 Life Science Roche GS junior. Amplicon Variant Analyzer (AVA 2.7) software  was used to align all read amplicons and to calculate variant frequencies at each nucleotide position relative to the HxB2 reference HIV-1 strain sequence. Raw data were submitted to GenBank under SRP033482 study accession number.To estimate the error rate of our UDS protocol, we calculated the percentage of errors generated from 2 steps of PCR (Platinum Taq Polymerase followed by FastStart High Fidelity polymerase amplification) and pyrosequencing of a plasmid control containing HIV-1 LAV reference HIV-1 strain (pNL4.3). The mean error rate was calculated using a triplicate experiment and the percentage of variations compared to the plasmid HIV-1 sequence. We estimated an overall mean error rate of 0.21±0.07% (mean reads per position: 3007±1477). Deletion and insertions events corresponded to 29.8% and 61.9% of the overall errors, with 0.07±0.06% of deletion and 0.12±0.07% of insertion relative to total reads. These values are in accordance with the rate of errors generated by the FastStart High Fidelity polymerase As input HIV RNA was not quantified, the expected sampling error depended on sample viral load. The stochastic effects of sampling variation were limited when the starting RNA copy number was higher than 10,000 copies/mL Drug resistant mutations (DRMs) were thus accepted as significant variants when present at a frequency of >1% among the total number of reads and in both strands.http://www.hivfrenchresistance.org) in order to determine the overall burden of resistance to antiretroviral (ARV) drugs and resistance to the prescribed treatment. The weighted Genotypic Sensitivity Score (wGSS) scores the resistance to the prescribed treatment and was calculated as the sum of the scores obtained for each prescribed ARV drug. In this system, ARV drugs are considered to display full, intermediate or null antiviral activity and scored as 1, 0.5 or 0 for PIs, 1, 0, 0 for NNRTIs and 0.5, 0, 0 for NRTIs Reverse transcriptase and protease DRMs were identified using SS and UDS at baseline and VF. Analyzed mutations associated with resistance to nucleoside reverse transcriptase inhibitor (NRTI), NNRTI and PI were referenced in the 2011 International AIDS Society (IAS)–USA drug resistance mutations list Paired t-tests were used to compare the mean number of DRMs detected by SS versus UDS for all DRMs, PI-DRMs, NRTI-DRMS and NNRTI-DRMs (Stata 12.1 software).10 copies/ml) were 248 (interquartile range IQR: 177–314) and 5.0 (IQR: 4.6–5.2) respectively. After a median duration of 14.3 months under therapy (IQR: 8.7–24.2), VF occurred in patients with a median of 3.7 log10 copies/ml of HIV RNA (IQR: 3.3–4.0).Twenty-nine patients achieving VF at least 6 months after initiating first-line ART had plasma samples available at baseline and at VF and were included in our study Table 2.Three patients were treated with a 2 NRTI-based regimen, 5 patients with 3 NRTIs, 5 patients with NRTIs + PI, 10 patients with NRTIs + boosted PI and 6 patients with 2 NRTIs + NNRTI. Co-prescribed PIs were Atazanavir, Lopinavir, Nelfinavir or Fosamprenavir and co-prescribed NNRTIs were Efavirenz or Nevirapine.10 cp/ml to 6.62 log10 cp/ml), and a median of 1739 (IQR: 1123–2585) reads per amplicon on viral HIV RNA from plasma collected at VF (VL range: 3.01 log10 cp/ml to 4.98 log10 cp/ml at failure). UDS of the protease region was successful for all of the 29 patients at failure and at baseline but RT UDS failed for 1 patient at baseline.Sequencing was performed at baseline on the last plasma sample available before ART initiation  and on the plasma sample corresponding to the date of VF. UDS performed on baseline viral HIV RNA yielded a median of 1663 (IQR: 1017–2533) reads per amplicon  of the patients and NNRTI-DRMs in 17.2% (5/29). UDS analysis showed that 51.7% (15/29), 3.4% (1/29), 10.7% (3/28) and 25.0% (7/28) of the patients had viruses harbouring additional low-frequency PI-, major PI-, NRTI- and NNRTI-DRMs respectively at baseline. The low-frequency NNRTI-DRMs corresponded to polymorphic Etravirine mutations  and Rilpivirine mutations (E138G) in a total of 4 and 2 patients respectively.These low-frequency DRMs could increase the overall burden of resistance to one or more ARV drugs in 4 of 29 patients (13.8%).Among the 21 patients with low-frequency DRMs, 8 had virus harbouring DRMs correlated with the future prescribed treatment. Nevertheless, the wGSS was not modified except for Patient 61 who harboured 2.1% of HIV variants with Y181C mutation that could imply resistance to its NNRTI treatment.Using Sanger sequencing, we determined that 20/29 patients had viruses harbouring DRM. Accessory PI mutations were detected in most of the patients but major-PI mutations were found in 17.2% of patients (5/29). NRTI- and NNRTI-DRMs were found in 58.6% (17/29) and 34.5% (10/29) of the patients respectively.Additional low-frequency PI-, major PI-, NRTI- and NNRTI-DRMs were found by UDS in 44.8% (13/29), 10.3% (3/29), 25% (7/28) and 21.4% (6/28) of the patients respectively. These low-frequency DRMs could increase the overall burden of resistance to one or more ARV drug in 9 of 29 patients (31%).Among the 18 patients with low-frequency DRMs, 12 had virus harbouring DRMs correlated with the prescribed treatment. These low-frequency DRMs detected only by UDS led to changes in susceptibility predictions to the prescribed treatment in 6/29 patients (20.7%), as shown by their VF UDS GSS lower than their VF SS GSS. Patients 14, 49 and 64 presented in their viral population 4.3% of M184I, 2.7% and 3.8% of M184V respectively that could be involved in resistance to the prescribed Lamivudine. Abacavir resistance was identified in Patient 46 who harboured 3% of viruses with L74V mutation and in Patient 86 who was infected with 34.9% of viruses carrying Y115F mutation. In Patient 40, 5 high-frequency PI-DRMs were found, leading to possible resistance to Lopinavir. The detection of 5% of variants with the additional Lopinavir resistance mutation L10V could explain an increase on resistance to this PI.Taking into account both high-frequency and low-frequency DRMs, 8 patients  had no resistance to their prescribed treatment, and 4 had even no DRMs related to their prescribed treatment Among the 38 additional low-frequency DRMs detected at baseline, 9 were related to the future prescribed treatment (8 patients). Among them, 1/5 PI-DRMs (Patient 23), 2/3 NRTI-DRMs (Patients 25 and 80) and 1/1 NNRTI-DRMs (Patient 61) were also detected at VF. All the PI-DRMs were accessory PI-DRMs. In the unique subject with baseline low-frequency NNRTI-DRM that could lead to resistance to the prescribed ART , both UDS and SS performed at virological failure showed the evolution of this mutation which was detected at high frequency (97.4%).Most of the treatment-related mutations found at VF were not detected at baseline by UDS. In 75% of the cases (21/28), VF was associated with the occurrence of NRTI DRMs, mainly the M184I/V mutation. Among the 26 patients receiving Emtricitabine (FTC) or Lamivudine (3TC), viruses harbouring the M184I/V mutation at VF were found in 14 patients by both SS and UDS and in 3 patients by UDS only. However, this M184 mutation was never detected at baseline by UDS. Pre-existing low-frequency TAM-NRTI mutations were found in 2/5 patients from whom high level of TAMs were detected at VF . Concerning the 4 patients with NNRTI-DRMs at VF, only one (Patient 61) had pre-existing low-frequency NNRTI-DRM at baseline. Among the 5 patients treated with PI-based regimen with viruses harbouring major PI-DRMs related to their treatment, none of these PI-DRMs was detected by UDS at baseline. Most of the polymorphic PI-DRMs were already detected at baseline by both SS and UDS.Our study gave new insights on the distribution of low-frequency variants harbouring PI-, NRTI- and NNRTI-DRMs present prior to first-line therapy and at virological failure, with emphasis on the evolution of these DRMs under ARV therapy. More than two-thirds of the patients presented additional low-frequency DRMs only detected by UDS, with 1.4-fold more DRMs detected by UDS at baseline and 1.3 fold more at VF. This increased prevalence of DRMs was in concordance with previous reports describing the abundance of low-frequency resistant variants , especially detected by UDS, on treatment-naive patients et al found a very low prevalence of M184V/I by UDS in chronically infected naive patients Twenty-six patients had a 3TC/FTC backbone in their regimen. The M184I/V mutation was detected in 17 of them at failure (only by UDS in 3 of them). Nevertheless, we never detected this mutation on samples collected prior to treatment even at low frequency. The absence of the M184V mutation in baseline circulating viruses is not in agreement with previous reports describing the detection of low-frequency variants carrying M184V mutations in acute One of the main questions sparked by these new ultrasensitive assays remains the significance and the clinical relevance of these low-frequency DRMs, and especially their role in subsequent virological failure.In our study, 4/29 patients had viruses harbouring low-frequency NRTI- or NNRTI-DRMs related to the prescribed treatment at baseline and these DRMs were found at high frequency at VF in 3 of them. Moreover, for the unique patient with viruses harbouring low-frequency DRM at baseline (Y181C at 2.1%) which may lead to a resistance to his future treatment by NNRTI, this mutation was found at high frequency (97.4%) by both UDS and SS at virological failure.The impact of low-frequency DRMs on the subsequent response to ARV likely depends on the percentage of low-frequency resistant variants and/or on the mutational viral load The impact on the virological outcome probably also depends on treatment regimen.Many studies clearly report a strong and significant association between low-frequency NNRTI-DRMs and a higher risk of virological failure in patients treated by NNRTI-based regimenThe few studies that evaluated the impact of low-frequency PI-DRMs on PI-based regimen found no associations with an increased risk of treatment failure Others studies have shown that low-frequency variants harbouring major PI-mutations are infrequently detected by UDS (most of them are accessory mutations with low Stanford HIVdb scores) and occurred in isolation in ARV-naive patients and in patients experiencing failure on PI boosted-regimen The very low prevalence of variants harbouring major PI-DRMs at significant levels combined with the high genetic barrier to resistance of PI could explain the fact that studies failed to show an impact of low-frequency PI-DRMs on virological response to boosted-PI based regimens. Further studies are needed to determinate if the existence of major PI-DRMs present at low frequency could affect the efficacy of PI/r containing regimens.Our work has several limitations.First, the ability to detect low-frequency variants depends on initial VL and on the amount of RNA copies used for RT-PCR. We were not able to quantify the number of RNA copies submitted to amplification procedure. Based on previously published data that consider RNA extraction efficiency, we assumed that the approximate number of templates derived from a sample with plasma HIV-1 RNA levels ≥4.5 log copies/mL and submitted to UDS was around 100–200 copies Secondly, patients were included if they experienced a VF on first-line therapy. The prevalence of low-frequency DRMs on this group of patients was not compared to a control group of patients with virological suppression. Hence, we could not evaluate the impact of the drug resistant low-frequency DRMs on virological outcome. Because of the small number of patients experiencing VF under a first-line ART for whom plasma samples were available both at baseline and VF, only 29 subjects were included in our study. Besides almost 45% of them received an antiretroviral therapy dating back from 2000 which is not state of the art anymore.Finally, adherence to HAART was not reported. Eight patients had no ARV drug resistance suggesting that their VF was related to non-adherence to treatment. Besides, most of the DRMs found at VF under first-line ARV therapy did not exist prior to treatment and were probably acquired through drug selection pressure, favoured by poor adherence or pharmacokinetic factors. Adherence to antiretroviral therapy is a major predictor of viral suppression and disease progression In conclusion, UDS identified significantly more DRMs than SS for each class of ARV , both in treatment-naive patients and in patients who experienced virological failure on first-line ARV. In few cases, these additional low-frequency DRMs may change the ARV susceptibility predictions to the prescribed treatment.Large prospective studies are now needed to assess the impact of these low-frequency DRMs on virological response, according to the proportion of each DRM and to the composition of the ARV regimen, before applying these ultrasensitive assays in routine clinical practice.


The study aimed to determine the prevalence, predictors, and self-reported management of HIV- or ARV-related symptoms among HIV patients prior to antiretroviral therapy (ART) and over three time points while receiving ART in KwaZulu-Natal, South Africa.A total of 735 consecutive patients  who attended three HIV clinics completed assessments prior to ARV initiation, 519 after 6 months, 557 after 12 months, and 499 after 20 months on ART.The HIV patients reported an average of 7.5 symptoms (prior to ART), 1.2 symptoms after 6 months on ART, 0.3 symptoms after 12 months on ART, and 0.2 symptoms after 20 months on ART on the day of the interview, with a higher symptom frequency amongst patients who were not employed, had lower CD4 cell counts, experienced internalised stigma, and used alcohol. The most common symptoms or conditions identified by the self-report included tuberculosis, diarrhoea, headaches, rash, nausea and vomiting, pain, neuropathy, lack of appetite, cough, and chills. Overall, the participants reported medications as the most frequently occurring management strategy, with the second being spiritual, and the third being complementary or traditional treatments. The use of all other management strategies decreased over the four different assessment periods from prior to ART to 20 months on ART.This study found a high symptom burden among HIV patients, which significantly decreased with progression on antiretroviral treatment. Several symptoms that persisted over time and several sociodemographic factors were identified that can guide symptom management. The utilisation of different symptom management strategies  should be taken into consideration in HIV treatment. Few studies, especially longitudinal, have described the self-reported symptoms of people living with HIV (PLHIV) and antiretroviral therapy (ART) patients in sub-Saharan Africa  recruited from the three public hospitals in the Uthukela health district in KwaZulu-Natal from October 2007 to February 2008. All ARV-naxve patients who were about to commence ARVs (18 years and above) and who consecutively attended the HIV clinics during the recruitment period were eligible for this study. Systematic sampling was used by asking health care providers for referrals of ART-naive patients (eligible for ARV treatment but who had not commenced ARV treatment yet). Physicians from the three selected public clinics asked every consecutively visiting ART-naive patient meeting the inclusion criteria of being 18 years or over if they would like to complete a confidential survey and interview concerning their health and social situation. This would include information from their medical records on details of their medical condition, laboratory tests, and treatment. It was made clear to patients that their participation in this study was voluntary and that a decision not to participate would not affect their medical care. If the potential participant indicated an interest in participating, the health care provider then referred them to an external Human Sciences Research Council research assistant. The ART-naïve patients were then asked to sign and complete a consent form before the interview took place in a private area in or outside the clinic. The interviews were conducted by four trained external HSRC researchers (one or two per HIV clinic) in interviewer-administered semi-structured interviews. Permission to access patient medical records was sought from both the patient and the health worker/manager. Questionnaires were anonymised, with no personal identifying information recorded on them. Recruitment took place over a period of four months, with a 97.8% participation rate. Data were collected using an interviewer-administered semi-structured questionnaire.This is a prospective study of all treatment-naxve patients (The questionnaire was translated into the major language spoken in the study area (Zulu) and verified by a second translator. Where inconsistencies were found, these were corrected . The patThe patients were interviewed with an anonymous questionnaire that requests information on sociodemographic characteristics, clinical history, and health-related characteristics and health beliefs. Clinical data relating to date of HIV diagnosis, HIV acquisition and transmission risk factors, current CD4 cell count, and viral load were obtained from the medical chart.α reliability coefficient of this 64-item scale was 0.95 at baseline and 0.84, 0.95, and 0.78 for the three subsequent assessment periods, indicating the excellent internal consistency reliability of the items. The presence of any symptom was added up to form the total HIV-related symptom burden.The SSC-HIVrev is a 72-item checklist of HIV/AIDS-specific physical and psychological symptoms scored using the following scale: 0 = not checked (not present today), 1 = mild, 2 = moderate, and 3 = severe . The valParticipants were asked to describe three to six HIV- and/or ARV-related symptoms they had experienced, list the strategies  they used to manage these symptoms, and rate the perceived effectiveness of these strategies  provided an overall adherence assessment for a longer time interval. The VAS is a valid method of assessing medication adherence . AdherenThe alcohol use disorders identification test (AUDIT-C) focuses solely upon the consumption of alcohol ) . Becausep-values lower than 0.05 were entered in the corresponding multiple regression model. A forward procedure based on the quasi likelihood ratio test produced the final model.Data were analysed using Statistical Package for the Social Sciences for Windows software application programme version 17.0. Frequencies, means, standard deviations, median, and interquartile ranges were calculated to describe the sample. To identify the pattern of factors characterising the HIV-related symptom burden at any assessment, linear regression models based on generalised estimating equations (GEEs) were used. These models allowed for the consideration of the correlation among within-subject repeated measures . For theA total of 735 patients (217 men and 518 women) completed a baseline questionnaire at Time 1 prior to initiating ART. Follow-up questionnaires were completed at the 6-month follow-up by 519 patients within this cohort (139 men and 370 women) who had now been on ART for 6 months, 12 months later by 557 patients within this cohort (157 men and 396 women), and 20 months later by 499 patients (126 men and 333 women) and 236 (32.1%) participants were lost to follow-up (including transfers): 83 (11.3%) were known to have died, 74 (10.1%) transferred care elsewhere, 14 (1.9%) refused participation, 12 (1.6%) were not initiated on ART, and 53 (7.2%) could not be traced. At Time 4, the HIV medications for 380 (76.3%) patients were Lamivudine (3TC), Stavudine (d4T) + Efavirenz and for 118 (23.7%) patients were Lamivudine (3TC), Stavudine (d4T) + Nevirapine.The mean age of the participants at baseline assessment was 35.9 years (SD = 9.7) and the educational level of the majority (81.0%) was less than Grade 12. Almost three-quarters (71.8%) had never married, 61.2% were unemployed and 20.2% employed, more than half (52.3%) had a child care grant, 29.9% had a formal salary, and 7.9% had no income. Almost two-thirds (63.3%) resided in a rural area, most (73.5%) had recently been (within the past year) diagnosed as being HIV positive, and almost all (96.1%) had disclosed their HIV status to someone. While baseline characteristics were similar between men and women, women were more likely to be younger and receiving social (including child care) grants and men were more likely to be married or cohabiting, employed, and have a formal salary. The median CD4 count at the 20-month follow-up was 446 cells/cu.mm compared with 261 cells/cu.mm at the 12-month follow-up, 130 cells/cu.mm at the 6-month follow-up, and 119 cells/cu.mm prior to ARV initiation. A study attrition analysis comparing participants who left the study with those who stayed found significant differences in terms of a lower educational level, rural residence, employment status , and lower HIV symptoms for those who stayed in the study and no significant differences in terms of gender, age, religion, income, time since HIV diagnosis, CD4 cell counts, internalised stigma, discrimination experience, and alcohol use see .F = 2193.69; p < 0.001). An examination of individual HIV-symptom items indicated that the percentage of patients scoring mild, moderate, or severe (defined as a score of 1 or more) decreased over time for all items. The most obvious change was for ‘concerned over weight loss’ and ‘dry mouth'; approximately one-third (35.7% and 31.7%) of patients scored ‘yes’ on these items at baseline, but none scored ‘yes’ at the 20-month follow-up. The five most frequently reported symptoms at Time 1 were concern over weight loss, headaches, dry mouth, memory loss, and weakness; at Time 2, chills, headaches, diarrhoea, concern over weight gain, and dry mouth; at Time 3, headaches, diarrhoea, numbness/tingling of legs, lack of appetite, and painful joints; and at Time 4, painful joints, diarrhoea, lack of appetite, fever, and headaches. The scores for each HIV-symptom item at each of the four assessments are given in The baseline mean total score on the HIV-symptom scale for the 735 patients was 7.5 (SD = 9.6), which significantly decreased at Time 2–1.2 (SD = 2.6), at Time 3–0.3 (SD = 2.1), and at Time 4–0.2 (SD = 0.9) (β coefficient 0.06 (95% CI: –0.04 to 0.201) (p = 0.091).To identify at any assessment the pattern of factors characterising the HIV-related symptom burden , linear regression models based on generalised estimating equations (GEEs) were used. When univariate analyses were employed and in the GEE multiple regression model see , not beiParticipants first listed 3–6 HIV- or ARV-related symptoms. The five most common symptoms or conditions identified at Time 1 (prior to ART) were tuberculosis, headaches, diarrhoea, weight loss, and cough; at Time 2, headaches, diarrhoea, rash, nausea and vomiting, and pains; at Time 3, headaches, numbness/tingling, pains, rash, and lack of appetite; and at Time 4, diarrhoea, rash, headaches, chills, and pains see .Regarding management strategies for the self-reported HIV- or ARV-related symptoms, medications were the most frequently mentioned care strategies and ranked first in overall use, ranging from 22.5% at Time 3 to 85.9% at Time 2. This was followed by spiritual care at 33.3% at Time 1 and at 46.4% at Time 2 and complementary or traditional treatments at 37% at Time 1 and at 30.9% at Time 2. Overall, all different management strategies decreased over the four different assessment periods from prior to ART to 20 months on ART see .In this study, HIV patients reported an average of 7.5 symptoms (prior to ART), 1.2 symptoms after 6 months on ART, 0.3 symptoms after 12 months on ART, and 0.2 symptoms after 20 months on ART on the day of the interview, with a higher symptom frequency amongst patients who were not employed, experienced internalised stigma, used alcohol, and had lower CD4 cell counts. The duration of HIV diagnosis was not associated with the HIV-related symptom burden. Other studies , this stThe five most frequently reported symptoms on the structured questionnaire at Time 1 were concern over weight loss, headaches, dry mouth, memory loss, and weakness; at Time 2, chills, headaches, diarrhoea, concern over weight gain, and dry mouth; at Time 3, headaches, diarrhoea, numbness/tingling of the legs, lack of appetite, and painful joints; and at Time 4, painful joints, diarrhoea, lack of appetite, fever, and headaches. In an open-ended question, the most common symptoms or conditions identified included tuberculosis, diarrhoea, headaches, rash, nausea and vomiting, and pains. Similar symptoms have been reported from other studies in Southern Africa . It is iOverall, the participants reported medications as the most frequently occurring management strategy and the most effective, second was spiritual care, and third was complementary or traditional treatments. This finding suggests that participants in this study used their medical provider as a major source of the management of their symptoms. Furthermore, spiritual, complementary, or traditional treatments apparently play an important role in PLHIV. All types of management strategies decreased over the four different assessment periods from prior to ART to 20 months on ART. In a different study in Southern Africa using the same management categories, medications were also ranked first, complementary treatments were ranked second, and spiritual care was ranked seventh . The useViral load data were only available for a few participants and so this category was therefore excluded from the analysis. Furthermore, the assessment of ART adherence and other measures was relied on the self-report. The study results may be biased in favour of those who survived and were healthy enough to participate in follow-up. Sample attrition is a methodological artefact that can potentially influence longitudinal studies . The symThis study found a high symptom burden among HIV patients, which significantly decreased with progression on antiretroviral treatment. This means that antiretroviral treatment is effective in reducing the HIV-related symptom burden among HIV patients.Several symptoms that persisted over time and several sociodemographic factors that were identified can guide symptom management using the existing WHO clinical guidance that emphasises the essential role of palliative care alongside HIV treatment.The utilisation of different symptom management strategies  should be taken into consideration in HIV treatment.


HIV integrase inhibitor use is limited by low genetic barrier to resistance and possible cross-resistance among representatives of this class of antiretrovirals. The aim of this study was to analyse integrase sequence variability among antiretroviral treatment naive and experienced patients with no prior integrase inhibitor (InI) exposure and investigate development of the InI drug resistance mutations following the virologic failure of the raltegravir containing regimen.Sequencing of HIV-1 integrase region from plasma samples of 80 integrase treatment naive patients and serial samples from 12 patients with observed virologic failure on raltegravir containing treatment whenever plasma vireamia exceeded >50 copies/ml was performed. Drug resistance mutations were called with Stanford DB database and grouped into major and minor variants. For subtyping bootstrapped phylogenetic analysis was used; Bayesian Monte Carlo Marcov Chain (MCMC) model was implemented to infer on the phylogenetic relationships between the serial sequences from patients failing on raltegravir.Majority of the integrase region sequences were classified as subtype B; the remaining ones being subtype D, C, G, as well as CRF01_AE , CRF02_AG and CRF13_cpx recombinants. No major integrase drug resistance mutations have been observed in InI-treatment naive patients. In 30 (38.5%) cases polymorphic variation with predominance of the E157Q mutation was observed. This mutation was more common among subtype B  than non-B sequences , p=0.00099, OR: 5.91 (95% CI:1.77-22.63)]. Other variants included L68V, L74IL, T97A, E138D, V151I, R263K. Among 12 (26.1%) raltegravir treated patients treatment failure was observed; major InI drug resistance mutations  were noted in four of these cases . Time to the development of drug resistance ranged from 2.6 to 16.3 months with mean increase of HIV viral load of 4.34 (95% CI:1.86-6.84) log HIV-RNA copies/ml at the time of emergence of the major mutations. Baseline polymorphisms, including E157Q were not associated with the virologic failure on raltegravir.In InI treatment naive patients polymorphic integrase sequence variation was common, with no major resistance mutants. In the treatment failing patients selection of drug resistance occurred rapidly and followed the typical drug resistance pathways. Preexisting integrase polymorphisms were not associated with the treatment failure. HIV integrase, being one of the key retroviral enzymes necessary for successful replication, is one of attractive targets in the treatment of HIV infection. Integrase inhibitors, targeting one of the essential steps of the virus life cycle, namely strand transfer have been approved for the clinical practice in 2008, and have proven to be highly efficient in treatment of both antiretroviral-naive and experienced individuals-6. As poDespite high efficacy observed among treatment experienced patients with drug resistance to other antiretroviral drug classes, low genetic barrier to resistance and possible cross-resistance among integrase inhibitors is a limiting factor in the practical use of these compounds. Virologic failure has been associated with major, signature mutations within the catalytic domain of the enzyme, and include Y143R/C, N155H Q148K/R/H integrase sequence variants associated with significant susceptibility reduction both to RAL and elvitegravir (EVG). AccumulIn this study HIV-1 integrase sequences from patients observed at the Department of Infectious Diseases and Hepatology Pomeranian Medical University, Szczecin, Poland and Out-Patient’s Clinic of Acquired Immunodeficiency, Regional Hospital, Szczecin Poland were obtained. Bioethical committee approval  was obtained for this analysis. Informed consent was provided and obtained from study participants. Eighty samples from patients who have never received the integrase inhibitors were selected, including forty-six pretreatment ones from individuals who have later received raltegravir (RAL). Additionally, sequences were obtained at the time of virologic failure on RAL , whenever such a failure have occurred. Virologic failure was defined as two consecutive viral loads >50 HIV RNA copies/ml. To assess adherence number of dispensed monthly doses of antiretroviral medication divided by the number of follow-up months, expressed as a percentage, was used.http://pssm.cfenet.ubc.ca). In the failing group estimated adherence ranged from the 58% to 99%. In four cases  major InI drug resistance mutations have been observed. Mean viral load on raltegravir treatment in the failing group was 2.79 (95% CI:2.35-3.22) log HIV-RNA copies/ml being notably higher among patients with developed drug resistance [mean of 3.73 (95% CI:3.19-4.28) log HIV-RNA copies/ml] compared to the failing ones without the InI drug resistance mutations [mean: 2.79 (95% CI:2.35 -3.22) log HIV-RNA copies/ml]  and p=0.38, OR 1.83 (95% CI:0.38-10.05), respectively]. In 14 cases with virologic success accessory mutations were present prior to the raltegravir introduction . Among virologically failing patients E157Q was noted in one patient with N155H mutant, in three E157Q variant was present at baseline and consistently in the sequences obtained on RAL therapy while in two patients either R263K or L74IL were present at baseline and disappeared in the subsequent sequences on virologically unsuccessful treatment.Development of drug resistance mutations followed two patterns: major N155H with or without subsequent accessory V151I, E92EQ, V151I, G163R mutants (three cases) and Q148H accompanied by G140S mutant (one case) Figure. To deteIt must be noted, that three of the patients with developed drug resistance were heavily experienced with reverse transcriptase (RT) and protease (PR) mutations , while one patient (number 3) due to endocarditis and kidney failure was treated with live-saving but suboptimal therapy which consisted solely of ritonavir-boosted saquinavir and raltegravir.de novo development of drug resistance mutations decreasing the susceptibility of HIV against this class of antiretrovirals to provide the virologists and clinicians with the current data allowing for adequate therapeutic strategies.Integrase inhibitors remain an attractive option and have become a vital component of the modern antiretroviral treatment, especially among patients with preexisting drug resistance or treatment complications,40-45. IIn the presented study, among the InI naive patients no major drug resistance mutations have been observed, however, accessory mutations have been common (38.5%). Of the noted variants, four  have previously been described as polymorphic, occurring in >1% of integrase sequences. These iIn the group of the RAL treated patients treatment was successful in 73.9% of cases, while the 8.3% of the failing patients developed major drug resistance mutations significantly reducing susceptibility to both raltegravir and elvitegravir. Number of virologic failures was higher than observed in the STARTMRK and BENCHMRK trials for the patients with high baseline genotypic scores,5,6,15, in vivo studies[Integrase drug resistance profiles in four treatment failing patients with drug resistance followed two typical pathways, Q148H/G140S (one case) and N155H/V151I (three patients), associated with high level resistance to raltegravir and cross-resistance to elvitegravir-18,61,62 studies. The N15 studies,67. This studies.In summary, the results of this study provide data for the clinical practice and the treatment with integrase inhibitors. No major mutations associated with integrase dug resistance have been found in the pretreatment samples, however the polymorphic variation was common. In the failing patients selection of drug resistance occurred rapidly, and followed the typical pathways with accumulation of the drug resistance. Poor adherence, preexisting drug resistance and suboptimal combination were the key reasons associated with the development of the drug resistance.The authors declare that they have no competing interests related to this study.Conceived and designed the study: MP. Performed the experiments and analyses: MP AU. Analyzed the data: MP. Contributed reagents/materials/analysis tools: MP AU. Statistical and phylogenetic analyses: MP. Wrote the paper: MP DB AU ABK. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/368/prepub


Predictors of immuno-virologic outcomes and discordance and their associations with clinical, demographic, socio-economic and behavioral risk factors are not well described in Nigeria since HIV viral load testing is not routinely offered in public HIV treatment programs.The HACART study was a multi-center observational clinic-based cohort study of 2585 adults who started HAART between April 2008 and February 2009. A total of 628 patients were randomly selected at 12 months for immuno-virologic analyses.3) was 77.4% and immuno-virologic discordance rate was 33%. In multivariate logistic regression, virologic failure was associated with age <30 years , anemia (Hemoglobin < 10 g/dl) , poor adherence , and post-secondary education . Immunologic failure was associated with male gender , and age <30 years . Virologic failure with immunologic success (VL-/CD4+) was associated with anemia , poor adherence , and post-secondary education .Virologic suppression rate (<400 copies/ml) was 76.7%, immunologic recovery rate (CD4 change from baseline ≥50 cells/mmAlthough favorable immuno-virologic outcomes could be achieved in this large ART program, immuno-virologic discordance was observed in a third of the patients. Focusing on intensified treatment preparation and adherence, young patients, males, persons with low educational status and most importantly baseline anemia assessment and management may help address predictors of poor immuno-virologic outcomes, and improve overall HIV program impact. Viral load testing in addition to the CD4 testing should be considered to identify, characterize and address negative immuno-virologic outcomes and discordance. Every HIV treatment program is confronted with the triple challenge of addressing gaps in coverage (access), retention in care and improving treatment outcomes. With HIV treatment access now a reality in most of sub-Saharan Africa (SSA), attention and concerns have rightly shifted to issues of retention in care, negative treatment outcomes, HIV drug resistance and treatment failures -4. TreatBy the time most patients are diagnosed with immunologic failure (using 6 monthly CD4 cell counts) or clinical failure , multiple drug resistance mutations may have developed compromising and complicating future drug treatment options ,9. FurthPrevious immuno-virologic outcomes studies in Africa -12 have The study was reviewed and approved from all the Institutional Review Boards (IRB) of all three index hospitals in Nigeria, the Nigerian National Health Research Ethics Committee (NHREC), and the University of Maryland School of Medicine, Baltimore, Maryland, USA. Written informed consent was obtained from all the study participants after counseling and introduction of the study.The HIV AIDS Care and Anti-Retroviral Therapy (HACART) study was a large multi-center observational clinic-based cohort study that described the predictors of loss to follow-up, immuno-virologic outcomes, immuno-virologic discordance and sub-optimal drug adherence within the AIDS Care and Treatment in Nigeria (ACTION) project. Funded by the U.S. Centers for Disease Control and Prevention Global AIDS Program (CDC GAP), the ACTION project is a tripartite partnership between the Institute of Human Virology of the University Of Maryland School Of Medicine, the Institute of Human Virology Nigeria, and the Nigerian Federal and State Ministries of Health.A cohort of 2585 initially ART naïve adults who started HAART between April 2008 and February 2009 were followed up for 12 months in three representative government hospitals in Nigeria: University of Abuja Teaching Hospital, Abuja (UATH), University of Benin Teaching Hospital, Benin (UBTH) and Asokoro District Hospital, Asokoro, Abuja (ADH). These sites consisted of a mix of tertiary (UATH and UBTH) and secondary (ADH) health facilities located in different regions of the country and serving different populations to increase generalizability. With 805(31%) lost to follow-up, a total of 628 out of the 1780 patients alive and active on the program at 12 months were randomly selected for in-depth interviews and laboratory work-up with detailed virologic and immunologic testing . The choice of HAART combination was both guided by national treatment protocols and the discretion of the attending physician but generally consisted of one of three first line regimens (Tenofovir (TDF) + Lamivudine (3TC) + Nevirapine (NVP) / Efavirenz (EFV), Zidovudine (AZT) + 3TC + NVP / EFV, Stavudine (d4T) + 3TC + NVP / EFV. Patients who were anemic were preferably given TDF instead of AZT and persons who were co-infected with tuberculosis were also preferably given EFV instead of NVP. EFV was generally avoided in women of child-bearing age especially if they were not adherent to double contraception methods. A three months’ supply of drugs were provided  with three monthly refill follow-ups and six monthly CD4 testing done together with adherence counseling sessions according to the national ART guidelines. Patients who default from scheduled clinic and pharmacy refill visits were followed-up by the home-based care team.Before commencement of ART most patients underwent detailed history, physical examination, treatment preparation, HIV disclosure counseling, symptomatic screening for tuberculosis and laboratory work-up. Laboratory tests done at baseline included CD4 cell count, complete blood count, liver function tests and serum creatinine test. There were some missing laboratory results due to laboratory reagent shortage, or administrative errors. Cotrimoxazole was provided to eligible patients whose CD4 counts were less than 200 cells/ml. HAART was provided if patients satisfied an immunologic criteria (CD4 less than 200 cells/ml irrespective of WHO staging)http://distancecalculator.globefeed.com/Nigeria_Distance_Calculator.asp). Since there were no validated data on socio-economic (SES) classifications in Nigeria, a quintile of class 1–5 was developed based on socio-economic scores calculated using 4 indicators commonly used in social epidemiology studies .Standardized patient management and monitoring forms and interviews were used to capture detailed clinical, laboratory and demographic data, some of which were extracted from the CAREWare health information management system. Adherence was measured objectively by pharmacy refill records and subjectively by self-report. Residential distance from treatment sites to closest known town was determined in kilometers using the official Nigerian national road distance calculator and a unique online Nigerian distance tool at Globefeed.com (-) was defined as a HIV RNA level of equal or more than 400 copies / ml at the 12 month visit in line with local laboratory assay detection limits  for HIV-1 RNA in human plasma using Roche Amplicor version 1.5; Roche Diagnostics, Basal, Switzerland) and national treatment guidelines in Nigeria. Even though baseline HIV RNA levels were not done, it was expected that with adherence to potent HAART regimen, viral load levels would generally be undetectable (< 400 copies/ ml) by 12 months. Fresh samples from UBTH were processed and tested on site and fresh samples from AUTH and ADH were processed and tested at the National IHV reference laboratory onsite at ADH. Immunologic failure (CD4-) was defined with the WHO definition of “fall in CD4 count to baseline (pre-HAART) or below baseline levels (zero CD4 cell change)” or failure of CD4 cell to improve by at least 50 cells at 12 months [-/CD4+) and immunologic failure with virologic success (VL+/CD4-).Virologic failure  were categorized for data analysis. Hemoglobin was categorized either as a dichotomous variable using hemoglobin cut-off of 10 g/dl for anemia. Kruskal Wallis and Pearson χreenland ,17. CovaAll results were assessed at the 5% level of statistical significance for a 2-sided test. We also compared baseline demographic and laboratory parameters among patients that were sampled at 12 months and those who did not make it to 12 months. Some of the viral load and CD4 samples (6% and 5% respectively) were missing or were lost at random due to laboratory and / or administrative error. All missing data was assumed missing completely at random (MCAR) and multiple imputations  method) using STATA did not show any differences in study result interpretation. All analyses were done with STATA version 11 .3 (IQR: 85–223), baseline median hemoglobin level was 10.9 g/dl (IQR: 9.3-12.4) and anemia based on hemoglobin less than 10 g/dl was common . Majority of the patients were married . HIV disclosure was high  and most of the patients  lived less than 50 kilometers from their respective treatment sites. More than half of the patients  were on a TDF-containing regimen at baseline with no substitutions within the 12 months. Even though low educational achievement levels with no formal education were seen in 301 (47.9%) patients, however, socio-economic indicators showed high employment levels .Figure There were differences in patients’ characteristics by site on all variables except marital status, HIV disclosure, adherence by self-report, smoking and drug use history. Males differed significantly from females in age at HAART initiation, marital status, TB history, baseline CD4 cell count, baseline hemoglobin count, employment status and smoking and alcohol history. This study was focused on outcomes among those alive at 12 months, and because of the random selection of patients, there were no major differences in the 628 patients selected for the study and the other 1152 who were alive at 12 months and not selected for the study (data not shown). There were differences in the baseline characteristics of age, residential distance from treatment site, baseline CD4 count, baseline anemia status and anti-retroviral drug regimen between the patients who were lost-to-follow-up and those alive and in the study at 12 months (Table 3 (IQR: 87–225) compared to those with viral load > 400 at 122 cells /mm3 (IQR: 76–196) . Of 596 patients with CD4 cell counts (32 samples were not usable due to laboratory mishaps or administrative error) the immunologic failure rate was 22.7%. While 60% (n=338) had ideal immuno-virologic success (VL+/CD4+), immuno-virologic discordance rate was 33% (Table -/CD4+) and 16% (n= 90) of the patients having had immunologic failure with virologic success (VL+/CD4-).Of the 591 with completed and validated viral load results, 453 had viral load results < 400 copies/ml giving a virologic suppression rate of 76.7% and thus a virologic failure rate of 23.4%. The median baseline CD4 cell count for patients with viral load < 400 copies / ml was 147 cells/mmIn the univariate logistic model Table , a decreA decreased odds of immunologic failure was not significantly associated with any predictor in the univariate or multivariate regression models Table . However-/CD4+ compared to VL+/CD4+ was significantly associated with poor adherence as monitored by pharmacy refill records  and anemia (Hemoglobin < 10 g/dl)  while decreased odds was associated with post-secondary educational achievement   and higher baseline CD4 > 200 (9.90: 95% CI: 2.79-35.10) , age, lower baseline CD4 cell count, poor adherence and high mortality ,50-52. IIn addition, our study results strongly suggest that viral load testing should be incorporated into routine patient care, both at baseline and at least every 6 months. Without viral load results, 16% (n=90) of our study population would be switched to a second line ART regimen on the basis of their CD4 count results even though they had sustained viral suppression. Conversely, 17% (n=94) of our study population who were eligible for second line ART regimen would have continued on first line therapy inappropriately, also based on their CD4 count result only. This confirms the results of many studies that CD4 cell count alone is a poor predictor of virologic and clinical failure and an unreliable guide to appropriate clinical management of HIV infected patients on antiretroviral therapy -55. ViraLimitations of this study include less than ideal follow-up time (12 months), issues with measurement error, missing data and possible administrative error in recording variables common in busy government hospitals in resource-constrained settings. We use unimputed data and assumed all missing variables were missing completely at random (MCAR) and confirmed this assumption by doing multiple imputations  method) which produced similar results. There were no baseline HIV RNA viral load results and all patients are expected to have high viral loads at baseline. However, we expect patients fully adherent on potent HAART regimen to be undetectable at 12 months after commencement of therapy. This study was conducted in non-pregnant adults and may not be generalizable to pregnant women and children. This study has some unique strengths including its clinic-based cohort design with validated immunologic and virologic results even though viral load testing were not routinely performed in this program. The selection of different levels of care and different locations serving different populations improved the generalizability of our findings. In addition, since Tenofovir is not a common first line drug in ART programs in Africa, these results could provide additional information on programs that predominantly use this drug in low and middle income countries.In summary, rapid HIV scale up has been hugely successful in Nigeria but challenges in patient retention and outcomes continue. With immuno-virologic outcomes known to worsen over time due to progressively poor adherence, pill fatigue and drug side effects, efforts should be intensified at baseline to identify patients who are likely to develop poor outcomes. Our study clearly shows that comprehensive baseline treatment preparation efforts needs to intensify adherence counseling, address anemia, male health seeking behavior, active case detection for opportunistic infections, affordable viral load testing, and regular CD4 cell count testing. In addition, younger patients (less than 30 years) and those with less than post-secondary education must receive priority attention in comprehensive treatment preparation and adherence support efforts. Future research efforts should be geared towards developing feasible and affordable routine viral load testing platforms and identifying whether operationalizing malaria and helminthiasis co-treatment will improve immuno-virologic outcomes. The role of sub-clinical or undiagnosed opportunistic infections in immuno-virologic discordance situations needs to be further evaluated.a This criterion was changed to CD4 count less than 350 cells / ml in 2010 in line with the WHO recommendation.3TC: Lamivudine; ABC: Abacavir; ADH: Asokoro District Hospital, Asokoro, Abuja; AIDS: Acquired Immunodeficiency Syndrome; ARV: Antiretroviral; ART: Antiretroviral therapy; AUROC: Area Under Receiver Operating Curve; UATH: University of Abuja Teaching Hospital, Gwagwalada; AZT: Azidothymidine / Zidovudine; BMI: Body mass index; CD4: Cluster of differentiation 4 T-helper cells; D4T: Stavudine; EFV: Efavirenz; FTC: Emtricitabine; Global Fund: Global Fund to fight AIDS, TB and Malaria; HAART: Highly active antiretroviral therapy; HACART Study: HIV AIDS Care and Antiretroviral Therapy study; HIV: Human immune-deficiency virus; IHV: Institute of Human Virology, Baltimore, Maryland; IHVN: Institute of Human Virology Nigeria; LPV/r: Lopinavir boosted with ritonavir; LTFU: Loss to follow-up; MAP: Multi-country AIDS Program of the World Bank; M&E: Monitoring and Evaluation; MOHN: Ministry of Health, Nigeria; MTCT: Mother-to-child transmission of HIV; NHREC: Nigerian Health Research Ethics Committee; NNRTI: Non-nucleoside reverse transcriptase inhibitor; NRTI: Nucleoside reverse transcriptase inhibitor; NtRTI: Nucleotide reverse transcriptase inhibitor; NVP: Nevirapine; OI: Opportunistic Infections; PEPFAR: President Emergency Fund for AIDS Relief; PI: Protease inhibitor; PLWHA: Persons living with HIV AIDS; PMM: Patient monitoring and management forms; TB: Tuberculosis; TDF: Tenofovir disoproxil fumarate; UBTH: University of Benin Teaching Hospital, Benin; UNAIDS: Joint United Nations Agency for AIDS; VL: HIV viral load; WHO: World Health OrganizationThe authors declare that they have no competing interests.Conceived and designed the study: CA, WB, CB, MC. Study implementation: CA, AA, EE, HO, SA, OA. Data analysis: CA, MC. Contributed reagents / materials / analysis tools: CA, AA, WB, MC. Wrote paper: CA. Reviewed paper: CA, MC, CB, AA, ME, WB. Supervised patient care: EE, HO, SA, ME. Coordinated ACTION project: WB, PD, MC, AA, ME. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/113/prepub


Replicative phenotypic HIV resistance testing (rPRT) uses recombinant infectious virus to measure viral replication in the presence of antiretroviral drugs. Due to its high sensitivity of detection of viral minorities and its dissecting power for complex viral resistance patterns and mixed virus populations rPRT might help to improve HIV resistance diagnostics, particularly for patients with multiple drug failures. The aim was to investigate whether the addition of rPRT to genotypic resistance testing (GRT) compared to GRT alone is beneficial for obtaining a virological response in heavily pre-treated HIV-infected patients.Patients with resistance tests between 2002 and 2006 were followed within the Swiss HIV Cohort Study (SHCS). We assessed patients' virological success after their antiretroviral therapy was switched following resistance testing. Multilevel logistic regression models with SHCS centre as a random effect were used to investigate the association between the type of resistance test and virological response (HIV-1 RNA <50 copies/mL or ≥1.5log reduction).Of 1158 individuals with resistance tests 221 with GRT+rPRT and 937 with GRT were eligible for analysis. Overall virological response rates were 85.1% for GRT+rPRT and 81.4% for GRT. In the subgroup of patients with >2 previous failures, the odds ratio (OR) for virological response of GRT+rPRT compared to GRT was 1.45 (95% CI 1.00-2.09). Multivariate analyses indicate a significant improvement with GRT+rPRT compared to GRT alone .In heavily pre-treated patients rPRT-based resistance information adds benefit, contributing to a higher rate of treatment success. Combination antiretroviral therapy (cART) has dramatically reduced HIV related morbidity and mortality. Potent new drugs for patients with multiple drug resistance have been introduced -5. NeverTwo technical principles are in use today for resistance testing: Genotypic resistance tests (GRT) and phenotypic resistance tests (PRT). GRT is based on population gene sequencing of defined DNA segments, typically to detect mutations, which represent at least 20% of the virus population and confer HIV-1 drug resistance ,11. As aPRT assesses viral expression. A special form of it, the replicative phenotypic resistance test (rPRT) utilizes several replication cycles of a recombinant infectious virus to follow viral propagation in the presence of antiretroviral drugs ,17. By pSeveral studies have demonstrated the clinical benefit and cost-effectiveness of GRT -25 compaIn the present study we included all available data of prospectively conducted resistance tests for patients enrolled in the much larger multicentre Swiss HIV Cohort Study (SHCS) and compared the virological outcome in patients initiating a new antiretroviral drug regimen based on results of either GRT alone or rPRT combined with GRT. The highly sensitive format of rPRT used in the SHCS allows the detection of less than 1% of resistant virus in a clinical sample with a mixed virus population . We therThe SHCS is a prospective cohort study with continuing enrollment of HIV-infected individuals aged 16 years or older . The SwiThe reason for resistance testing has to be provided by the clinician ordering a given resistance test. The specified categories for resistance testing are: drug naive prior to initiation of first therapy, primary infection, suspicion for resistant virus transmission, pregnancy, and drug failure. The indication "primary infection" is specified by characteristics of very early infection stages with skin rash, very high virus load and incomplete immunoreactivity; "resistant virus transmission" is indicated when high promiscuity or the involvement of highly therapy-experienced individuals is suspected. When the reason for testing was missing, we utilized information from the SHCS to classify patients. Individuals were considered to have had testing for drug failure if they had either RNA >1000 copies/mL, 1-2 previous ART regimens and RNA between 500-1000 copies/mL, or were on a salvage therapy (>2 previous ART regimens).GRT is performed in Switzerland in four dedicated laboratories of the SHCS that use different techniques ,34. One The rPRT system used in Switzerland is based on a position-precise ligation of patient-derived PR/RT sequences into a replication competent background of a standardized reference HIV-1. As the entire amplified virus population is retained during DNA plasmid propagation this process represents to the best extent possible the virus population present in a patient's blood at the time of blood draw. The subsequent introduction of the DNA plasmids into susceptible human reporter cells initiates a rapid HIV infection. The diagnostic system, termed deCIPhR/PhenoTecT, allows the reconstituted HIV to undergo in a time window of four days 3-4 rounds of replication in the presence of each drug separately. A first replication round in this system thereby eliminates any susceptible wild type viruses, while relevant drug resistant variants are amplified during several cycles. A stably integrated LTR-driven reporter is activated by HIV Tat, and its expression has been shown to directly correlate with cellular HIV infection . The deCThe primary endpoint of the study was virologic response defined as either HIV-1 RNA viral load <50 copies/mL or a reduction in viral load of ≥1.5 log copies/mL. Once an individual started the new cART regimen, any further regimen switches prior to achieving virological response were defined as a failure unless no HIV-1 RNA was measured.10 transformed), nadir CD4 cell count , number of previous cART regimens, cART regimen class, calendar year and adherence to antiretroviral drugs (maximum number of self-reported missed cART-doses in the 4 weeks prior to a cohort visit) [Our main predictor was the type of resistance testing an individual received: GRT alone or GRT plus rPRT. The following covariates were considered for inclusion in the analysis to adjust for potential confounding: age , gender, current intravenous drug use (IDU) or participation in a drug maintenance program, HIV-1 RNA (logt visit) .Baseline characteristics of the eligible population were summarized overall and by resistance test. We explored whether rPRT in addition to GRT was associated with higher rates of virological response. To study the effects of the type of resistance test on the success of therapy, multilevel logistic regression analysis was performed. SHCS centre was included in the model as a random effect to account for the potential higher correlation in response among individuals seen at the same centre.Based on our hypothesis that the benefit of rPRT would be greatest in those with previous drug failure, we pre-defined two subgroups for additional analysis: patients having a resistance test after any treatment failure and patients having a resistance test after >2 previous treatment failures.The association between explanatory variables and treatment success were assessed by odds ratios (OR) and 95% confidence intervals (CI); OR above 1 indicate that a covariate is positively associated with the outcome. All analyses were done with SAS 9.1 . The manuscript was written to comply with STROBE  guidelines .For the period 2002-2006 we identified 2268 individuals with a total of 2889 resistance test samples. Of these, 1459 tests from 1204 individuals were excluded. The reasons for ineligibility were no change of cART following resistance testing (49.0%), a change of cART later than one year following resistance testing (36.1%), patients being off cART for more than 6 months following resistance testing (8.6%), and 6.7% with no available HIV-1 RNA viral load following resistance testing , testing for transmission of resistant viruses in naïve patients (28.5%), pregnancy (3.5%), and unknown (1.5%). There was no relevant difference in the distribution of the indication for resistance testing according to the type of resistance test  in the GRT group and 85.1% (n = 188 of 221) in the combined GRT plus rPRT group achieved the primary endpoint of virological response (either VL <50 copies/mL or 1.5 log reduction). The type of success achieved did not vary by type of resistance test with 51.4% of those with GRT and 49.5% of those with GRT plus rPRT achieving a VL <50 copies/mL. Success rates for GRT and GRT plus rPRT in the subset with resistance testing due to failure were 74.4% and 79.7%, in salvage patients 69.0% and 77.5%, respectively. The OR in univariable multilevel logistic regression analysis for virological response of GRT plus rPRT compared to GRT was 0.85 (95% CI 0.59-1.24) and for the pre-specified subgroups of patients with any and >2 previous drug failures were 1.16 (95% CI 0.73-1.82) and 1.45 (95% CI 1.00-2.09), respectively  (Table The new potent drugs such as darunavir and etravirine were not yet marketed in Switzerland. Nevertheless, calendar year was considered as a possible confounder in the model. Yet it was not found to be a relevant variable. When adding it to the multivariable model in Table In this multicentre cohort study of prospectively assessed HIV-1 drug resistance in patients the addition of rPRT to GRT as compared to GRT alone showed a trend towards improved success rates for treatment with increasing levels of antiretroviral pre-treatment. In the subgroup of heavily pre-treated patients with multiple drug failures the addition of rPRT significantly improved virological outcome with a 70% increased odds for achieving treatment success after adjusting for confounders and SHCS centre. The clinical benefit of resistance testing must be critically evaluated in its clinical context. Between 1999 and 2007 resistance declined overall in the SHCS . This deFrom a virologist's point of view the add-on benefit of rPRT is of particular relevance in patients with multiple drug failure and archived mutations. In patients with multiple virological drug failure and multiple therapy changes the genomic complexity of deposited HIV sequences increases. Growing resistance coincides with a rise of viral quasispecies ,39,40. AOne intrinsic potential limitation of this study lies in the fact that the choice of requesting GRT or GRT+PRT was largely centre-dependent, thereby introducing a possible centre bias and depending on any centre's preference for certain regimens. However by using a multilevel or hierarchical model, the effect of resistance testing was estimated after adjusting for the measured or unmeasured effect of centre.Our study has several strengths. We used stringent and very conservative criteria to define the target population of this observational cohort study. The cohort represents an unselected population of HIV-infected individuals, which is larger than the populations included in previously published observational studies and clinical trials. In addition this study includes a relatively large number of females and IDU making it more representative. We were able to include important variables in our analysis known to be related to virological outcome. For example, our data indicate that the study population included a relatively large group of patients with adherence problems in comparison to the general patient population in the SHCS. Roughly one third of patients had indicated that they had missed more than 2 doses in the previous four weeks and one fifth of patients stated to have missed more than 2 consecutive doses. Thus, our findings should be interpreted in the context of a patient population that poses real challenges for optimal clinical management and most likely makes it more difficult to demonstrate an add-on benefit of rPRT to GRT than one would have seen in a clinical trial with a more selected patient population.one single virtual virus genome as done by GRT, and thus gives more conservative estimates of antiretroviral drug resistance. In contrast, GRT in such patients leads to over-simplification by indicating cross resistance patterns per viral genome that tend in reality to be more complex. As a consequence, GRT may in these patients over-interpret the viral resistance and erroneously indicate to clinicians and their patients a lower number of remaining treatment options. The higher percentage in the GRT group of PI containing regimes, paralleled by a reduction in NNRTI-containing ART combinations suggests two likely reasons: on the one hand a centre effect for the favoured therapy-scheme, on the other, due to their low genetic barrier, the prompt stop of NNRTIs after virological failure. This study did, however, not assess whether or not this decision was always based on the formal demonstration of predominant NNRTI-related resistance mutations.High molecular diversity of HIV is a common problem in long-term treated, highly therapy experienced patients. In such patients with complex resistances rPRT is able to assign resistances to several co-existing viruses rather than placing the gene mutations onto Previous studies on GRT and PRT have investigated virological outcome with mixed findings, either resulting in non-significant gains -21 or inEvidence from clinical trials investigating whether GRT, PRT or the combination of both improve virological outcome is limited. Subgroup analyses from trials suggest that PRT may improve clinical outcome in patients with multiple previous failure. Our findings are in line with those trials. Our study shows that rPRT, when added to GRT, may indeed lead to improved virological outcome, particularly in the population of heavily pre-treated patients. This is corroborated by the finding that a therapy status "no treatment at time of testing" is significantly less frequent for the GRT + rPRT group. This indicates that GRT + rPRT was more often chosen in complex therapy situations. As scientific basis: replicative PRT functionally dissects resistant virus populations and may reveal remaining viable regimens, particularly in patients with limited options and thereby increase the chance for virological success. In contrast GRT tends to place for analysis all mutations on one viral "consensus" genome.Our study suggests that a stepwise testing strategy adding replicative PRT for patients with multiple drug failure provides benefit for better clinical decision-making. Further studies are needed to confirm whether this strategy translates into improved virological outcome in patients with limited treatment options.The authors declare no competing interests. During the study period Th. Klimkait was part-time employee at InPheno AG, Basel.JF and TK conceived the study, participated in its design and coordination and wrote the manuscript. TG and HB carried out the statistical analysis and were also involved in the main writing process of the manuscript. SL and FH were responsible for the performance of the genetic and phenotypic laboratory resistance test analysis. HH, VW, JB, SY, PB, MC, CF, BH, PV, GL, EB, HG and MB were involved in clinical and laboratory data collection in their respective clinical centres and in interpretation of the data and participated in the review of the final manuscript.


Although stavudine (D4T) remains frequently used in low-income countries in Asia, associated long-term toxicity data are scarce. The aim of this study was to determine the long-term incidence of severe D4T-toxicity (requiring drug substitution) and associated risk factors in HIV-infected Cambodians up to six years on antiretroviral treatment (ART).This is a retrospective analysis of an observational cohort, using data from an ART program with systematic monitoring for D4T-toxicity. Probabilities of time to D4T substitution due to suspected D4T toxicity (treatment-limiting D4T toxicity) were calculated, a risk factor analysis was performed using multivariate Cox regression modelling. Out of 2581 adults initiating a D4T-containing regimen, D4T was replaced in 276 (10.7%) patients for neuropathy, 14 (0.5%) for lactic acidosis and 957 (37.1%) for lipoatrophy. The main early side effect was peripheral neuropathy (7.0% by 1 year). After the first year, lipoatrophy became predominant, with a cumulative incidence of 56.1% and 72.4% by 3 and 6 years respectively. Older age  and lower baseline haemoglobin  were associated with the occurrence of neuropathy. Being female , a higher baseline BMI , and TB treatment at ART initiation  increased the likelihood of lactic acidosis. Lipoatrophy was positively associated with female gender , an older age , and a CD4 count <200 cells/µL .Stavudine-based treatment regimens in low-income countries are associated with significant long-term toxicities, predominantly lipoatrophy. Close clinical monitoring for toxicity with timely D4T substitution is recommended. Phasing-out of stavudine should be implemented, as costs allows. HIV is one of the major health problems in low and middle income countries (LMIC), with over 30 million of people infected. Over the last several years, a successful scaling-up of antiretroviral treatment (ART) has occurred, with currently over five million individuals on treatment. Of these, around 14% live in Asia Given increasing reports of D4T-related toxicity, including neuropathy, lactic acidosis and lipoatrophy, WHO 2006 guidelines have recommended to use alternative drugs in stead of D4T Despite the ongoing use of D4T, its recognized potential of toxicity and the maturing of many ART programs We conducted a retrospective cohort study between March 2003 and December 2010 at the Sihanouk-Hospital-Center-of-Hope (SHCH) in Phnom Penh, Cambodia. Since March 2003, this tertiary care hospital provides comprehensive HIV care at no cost, as part of the national ART program. All adult, ART-naive HIV-infected patients having initiating D4T-based ART at SHCH were included.Treatment initiation on ART was according to WHO recommendations: all patients with WHO stage IV, WHO stage III with CD4 cell count <350 cells/µL or with CD4 cell count <200 cells/µL were eligible for ART. First line treatment consisted of a generic FDC containing D4T, lamivudine and nevirapine. Zidovudine or efavirenz was prescribed in case of contraindications to D4T or nevirapine. From 2006 on, D4T was prescribed as 30 mg bid, irrespective of body weight. Prior to that, dosing was done according to body weight with a higher dose (40 mg bid) for individuals with a body weight above 60 kg Before ART initiation, extensive patient counselling was done, including on the occurrence of toxicities. Patients were seen at monthly visits during the first six months after ART initiation. Subsequently, visits were scheduled less frequently (every 2–3 months) for clinically stable patients. All medical care was provided by physicians, supported by a team of nurses and adherence counsellors. At every clinical encounter, key issues were systematically addressed, including the clinical evolution, evaluation of treatment response and ART-related toxicity. Adherence was evaluated by pill counts at every visit, and using the visual analogue scale (VAS) every six months. Baseline laboratory testing included haematology, liver function tests, hepatitis B/C serology and CD4 cell count determination (FACSCount (Becton Dickinson). After ART initiation, a full blood count and CD4 cell count was done every six months. During the first months of ART, liver function tests were performed more regularly. In case of suspicion of treatment failure, a viral load test was done. Clinical and immunological criteria were used to guide indications for viral load testing. Cotrimoxazole prophylactic treatment was given for all WHO stage II/III/IV patients and all those with a CD4 count <200 cells/µL. All patients with WHO stage 4 disease or a CD4 count <100 cells/µL were started on fluconazole primary prophylaxis. Patients not presenting at their scheduled visit were contacted by phone. Those living in the neighbourhood of the hospital were visited at home. Patients not presenting at the hospital for a period of 6 months without additional information were defined lost to follow-up (LTFU). Additional program details and outcome data of the antiretroviral treatment program in SHCH have been published before All patients on D4T-containing regimens were routinely screened for associated toxicity using a standardized approach. At every clinic visit, patients were assessed for symptoms suggestive of symptomatic hyperlactatemia or lactic acidosis. Lactate level was measured for suspected cases only. Since no pH determination could be done, cases of symptomatic hyperlactatemia (SH) and lactic acidosis (LA) could not be differentiated and were grouped together as SH/LA. A case of SH/LA was defined as a patient on ART, presenting with compatible symptoms with other mimicking conditions ruled out, and a lactate level ≥2.5 mmol/L . D4T was systematically replaced for these patients. Up to 2005, no lactic acid levels could be measured and diagnosis was clinical. Neuropathy was assessed clinically by the HIV physician, D4T was substituted in case of WHO grade III/IV severity. Patients were assessed for lipoatrophy  at every encounter by self-report and clinical assessment. Decisions to substitute D4T were guided by the clinical severity of lipoatrophy combined with the patient's preference and perception of the body changes. Consequently, stavudine was essentially replaced for reasons of lipoatrophy if 1) clinically severe or 2) patients perceived this as severe and disturbing, even though clinically it was defined as less pronounced.Clinical and laboratory data were prospectively collected on a daily basis, using standardized data collection tools and stored in a database. Quality control of the stored data was done at regular intervals.P-values<0.05 in the final model. Data analysis was done using STATA version 11. The level of significance was set at P<0.05.The primary outcome was time to D4T substitution due to suspected D4T toxicity. We will refer to this in the text as treatment-limiting (or severe) D4T toxicity. The cumulative incidence of D4T substitution due to suspected toxicity was calculated using the Kaplan Meier methodology. Patients were censored at the date of D4T substitution, at the last visit for patients that died, were transferred out or were lost to follow-up, and at December 31, 2010 for the remainder. Patients switched to non-D4T containing second line regimens due to virological failure were censored at the date of switching and defined as having experienced no D4T-related toxicity event. A risk factor analysis was performed using multivariate Cox regression. Collinearity between variables was assessed. We used a back-ward selection method, retaining those variables with Since the launch of the HIV care program, clinical data have been routinely collected for purposes of program monitoring and evaluation, and research activities. Patients were requested to give written informed consent to store and use the data. No linkage of these data with other sources was done. The data collection and informed consent procedure were approved by the Institutional Review Board ITM  and the Institutional Review Board SHCH . No patient identifiers were included in the dataset used for this analysis.By December 2010, 2581 adult patients on D4T-containing regimen were included in analysis, with a median follow-up time of 1.3 years. Of these, 1341 (52%) were female, the median age at the start of treatment was 35 years. Eighty percent were in WHO stage III/IV at treatment initiation. The median baseline CD4 count was 87 cells/µL. The most frequent ART regimen was D4T/lamivudine/nevirapine, with 72% of patients initiating this regimen. In total, 686 (27%) of the patients were on TB treatment at ART initiation .Out of 2581 adult patients initiating a D4T-containing regimen, D4T was replaced in 276 (10.7%) patients for suspected D4T-related neuropathy, 14 (0.5%) for SH/LA and 957 (37.1%) for lipodystrophy. All reported cases of SH/LH were confirmed by lactic acid determination. The main early side effect was peripheral neuropathy (7.0% by 1 year), with a cumulative incidence of 16.6% and 19.0% by 3 and 6 years respectively. SH/LA was mainly seen after the first six months but remained rare overall, with a cumulative incidence of 1% by 6 years. After the first year, lipoatrophy became the predominant side effect, with a cumulative incidence of 56.1% and 72.4% by 3 and 6 years respectively .2), and low baseline haemoglobin (<10 g/dL) were associated with increased risk of D4T-associated neuropathy (2), use of efavirenz and being on TB treatment at ART initiation were associated with increased risk of SH/LA. Lipoatrophy was positively associated with older age and female sex. An increased risk was observed with low baseline CD4 cell count (<200 cells/µL) and low baseline haemoglobin. In multivariate analysis, neuropathy remained associated with older age and low baseline haemoglobin. Being female, overweight (BMI>25 kg/m2), and on TB treatment at ART initiation increased the likelihood of SH/LA. Lipoatrophy was positively associated with female gender and older age. A reduced risk was seen for patients initiating ART with a baseline CD4 count >200 cells/µL , low baseline body mass index (BMI)  before revision of the guidelines in 2006. D4T drug changes were driven by suspicion of D4T toxicity by the clinician, hence not necessarily with formal proof of D4T as the culprit drug. Reported data merely demonstrate associations, and not causation. Moreover, this program was probably better resourced with more intensive monitoring compared to other field settings, and this could possibly have resulted in over diagnosis of toxicities. With increasing experience of the program, diagnosis may have been influenced by negative perceptions about D4T by physicians and patients. However, patients were systematically evaluated at each clinical visit, with standard clinical assessment, patient management and reporting. We also note that data were collected prospectively using standardized data collection tools, with continuous monitoring of data quality and clinical management practices. Still, it remains that the unavailability of technical investigations to more rigorously diagnose the different toxicities could have led to misclassification. Moreover, at the start of the program, no lactic acid determination could be done. For lipoatrophy, diagnostic tools such as DEXA-scanning might have allowed to define the exact incidence of lipoatrophy over time. However, our main research question was not to define what happens ‘biologically’, but rather ‘operationally’ ie to what extent and for what reasons D4T is replaced over time within a program with close toxicity monitoring.D4T-based treatment regimens in low-income countries are associated with significant long-term toxicities. With seven out of ten patients developing lipoatrophy by six years of treatment, lipoatrophy represents the major long-term side-effect. Until D4T has been phased-out completely, close monitoring for toxicity combined with the integration of the patient's perspective and a low threshold for D4T-replacement is recommended.