Musculoskeletal disorders is an inflammatory, degenerative diseases and disorders that cause pain and functional impairments. Musculoskeletal disorders are common and the major global health concern among people with human immunodeficiency virus/acquired immunodeficiency syndrome which causes physical disability. Despite, it is a recognized health problem among human immunodeficiency virus-positive patients, there is a lack of data on musculoskeletal disorders among patients following anti-retroviral therapy in sub-Saharan Africa, particularly Ethiopia. Therefore, the main aim of the study was to assess the prevalence and associated factors of musculoskeletal disorders among adult human immunodeficiency virus-positive patients following anti-retroviral therapy.An institutional-based cross-sectional study was conducted from September 1st to October 1st, 2021 at University of Gondar Comprehensive Specialized Hospital, Gondar, Ethiopia. The data was collected through an interview-administered questionnaire and patient medical record review of 324 participants. Binary logistic regression was used to identify associated risk factors of musculoskeletal disorders. The strength of the association was detected by the adjusted odds ratio and P-value.The annual prevalence of musculoskeletal disorders among participants was 158 (48.5%) with , opportunistic infection , type of ART medication used, CD4-count , and change in anti-retroviral therapy regimen change  were significantly associated with musculoskeletal disorders.The prevalence of musculoskeletal disorders was moderate. Recent CD4 count, opportunistic infection, antiretroviral therapy regime at initiation, and anti-retroviral therapy regime change were significantly associated with musculoskeletal disorder. A multidisciplinary approach is required for preventing and treating musculoskeletal disorders among human immunodeficiency virus-positive patients following anti-retroviral therapy. Musculoskeletal disorders (MSDs) encompass a variety of conditions caused by inflammatory, degenerative, genetic and autoimmune diseases that affect the muscles, tendons, ligaments, joints, peripheral nerves and supporting blood vessels in the body , 2. MuscThe introduction of ART  in the late 1990s substantially increased the average lifespan of persons living with HIV/AIDS (human immunodeficiency virus/acquired immunodeficiency syndrome). The majority of people who follow the recommended ART protocol achieve immunological improvement and have a normal life expectancy –11. WithThe experience of living with HIV/AIDS also brings serious consequences to the patient’s quality of life and permeates all areas of life through anxiety, depression, stress, changes in sleep patterns, the breakdown of social and emotional relationships, and difficulties regarding sexuality. Because the environment can influence this dynamic, the experience of living with HIV/AIDS in places with different social, political, and economic conditions may also be factors that define the quality of life of these patients –23. DiffDespite the high incidence of MSDs among HIV/AIDS patients following ART, there is paucity of data on the prevalence and associated factors of musculoskeletal disorder among HIV/AIDS following ART in sub-Saharan Africa particularly in the study area, Ethiopia. Finding from this study have a significant impact to design an appropriate treatment and prevention strategies to reduce the disabling effect of MSDs among people living with HIV/AIDS. Therefore, the aim of this study was to determine the prevalence and associated factors of MSDs among adult HIV/AIDS patients following ART clinic at University of Gondar comprehensive specialized hospital, Gondar, Ethiopia, 2021.An institutional-based cross-sectional study was conducted from September 1st to October 1st 2021 at University of Gondar comprehensive specialized hospital, Gondar, Ethiopia. Gondar city is found in Amhara regional state,727km away from Addis Ababa the capital city of Ethiopia. According to the 2015 census conducted by the central statistical agency of Ethiopia, Gondar had a total population of 206,987(29). University of Gondar comprehensive specialized hospital is the one and only teaching referral hospital in Gondar city, which provides service for more than 5 million people. The HIV care services of the hospital were established in 2003 and have three clinics that includes adult ART, pediatrics ART, and Volunteer Counseling and Testing (VCT). A total of 1950 adult HIV-positive patients were following ART clinic at University of Gondar Comprehensive Specialized Hospital per month.All HIV- positive adult patients who were following ART clinic at University of Gondar Comprehensive Specialized Hospital were study’s source population.The study population included all HIV- positive adult patients who were following ART clinic at University of Gondar Comprehensive Specialized Hospital during the data collection period.This study included all selected HIV- positive adult patients aged at least 18 years who attended adult ART clinic and who were able to give informed consent.Adult HIV-positive patients who seriously ill and unconscious, women patients who were pregnant, and those who had trauma or surgery of the past 6 months prior to data collection at any part of their body were excluded from the study.The sample size was determined using single population proportion formula with assumptions of 95% confidence interval, 5% margin of error, and by taking anticipated prevalence of MSDs 50% since there were no studies in Ethiopia and sub-Saharan Africa. The Z-value of 1.96 was used at 95% CI. .Using correction formula for less than 10,000 population.Where N = source population, Nx = sample size for unlimited population and n = corrected sample size. Adding 10% nonresponse rate the final sample size for the first objective becomes 354.An odds ratio for the relationship between alcohol intake, presence comorbidity condition, gender and pain among HIV-positive patients were taken from previous study  to deterTherefore, the final sample size of the study was 354.Systematic random sampling was used to enumerate the study subjects, “K” was calculated to be 5, . Allocating randomly from 1 to 5 and by drawing with lottery method to determine the starting unit (3) then after arranging the patients based on the order of coming to the clinic, each study participant was selected every 5 intervals.Musculoskeletal disorder (yes/no).age, sex, religion, marital status, educational level, salary and ethnicity.BMI, Physical exercise, alcohol drinking and cigarette smoking.Total duration since HIV/AIDS confirmed, baseline CD4 count, baseline WHO clinical stage, WHO clinical stage at ART initiation, recent WHO clinical stage, opportunistic infections, total time of using ART drug, and ART regime at initiation.if the patient experiences a complaint of unpleasant sensation(pain), ache, discomfort interfering with their activity of daily life at any part of their body region at any time during the last twelve months .weight in kilogram divided by the square of the height in meters (kg/m2) underweight < 18.50, normal 18.50-24.99, overweight > = 25 .Someone taking any kind of alcohol more than about two bottles of beer at least twice per week .-Doing regular moderate to vigorous physical activity of 30 to 45 min duration 3 to 5 days per week.-Someone who smokes cigarettes in the last time and currently smokes more than two days or every day per week.Face to face interview was deployed to assess the patient’s background information, which consist socio-demographic and socioeconomic questions. Questionnaires were developed from different literatures. Patient’s clinical and laboratory information  was collected by reviewing of patient charts.The study participant’s musculoskeletal disorder was assessed by using Nordic Musculoskeletal questioner to evaluate musculoskeletal symptoms. The questionnaire was designed to determine the prevalence of musculoskeletal issues in a certain population while also considering where they occur on the body. The questionnaire had three sections: Section I: - socio-demographic, section II: -personal and lifestyle characteristics, and section III: - clinical and treatment characteristics .p < 0.05 in the final logistic model were considered statistically significant and the strength and direction of association were measured by adjusted odds ratio (AOR) with corresponding 95% confidence interval. Model fitness was checked through Hosmer–Lemeshow goodness of fit test and it was fitted.Descriptive statistics were obtained, and the results were provided in the form of frequency, percentage, mean, and table. The connection of independent variables with MSDs among adult HIV-positive patients was demonstrated using bivariate and multivariable logistic regression analysis. To identify the variables attributed with the outcome variable, associations among independent variables and dependent variables were initially examined using bivariate logistic regression analysis. The prospective candidate variables having a p-value of 0.2 in the bivariate analysis were then incorporated into the multivariable analysis. Finally, variables with The questionnaire was first prepared in English and translated into the local language Amharic and back translation to English to ensure conceptual integrity. Data were collected by four (BSc physiotherapists) and one supervisor (MSc physiotherapist). Both the data collectors and the supervisor were trained for one day on the objective, methodology, the importance of privacy, ensuring the confidentiality of the respondents and data collection approach of the research prior to the actual data collection. Before the data was officially collected, the questionnaire was pre-tested out of the study area on 5% (18) of the total sample size among the ART users in Bahir Dar city at Felege Hiwet referral hospital. Discussion was held and changes made based on the findings of the pre-test like making questions clear to the patients. The supervisors made routine checkups for completeness and consistency of the data. The questionnaire was reviewed and checked for completeness, accuracy and consistency by the supervisors and investigators to take timely corrective measures.Three hundred fifty-four HIV/AIDS patients at the ART clinic were selected for the study yielding a response rate of 324 91.5%). Among the participants, the majority were female 208(64.2%), in the age range of 25–44 years (48.5%), and most were married 290 (58.6%)). The income statuses of the participants were mean of 2623 birr (SD ± 0.87) and the majority had an income > 2000 birr (39.8%), completed secondary school 96(29.6%)), and regarding religion 259(79.9%)) were Orthodox. See  were nonsmokers and 311 (96%) were nonalcoholic. Regarding physical exercise, the majority 237(73.1%) were not doing physical exercise and most of them had a normal BMI of 212(65.4%). See (Table About 65.1% of the participants had HIV/AIDS for about greater than 9 years. The majority of the participant had baseline CD4 cell count 200–350(47.2%), CD4 count at ART initiation 200–350(47.5) and the recent CD4 count was 611 cell/mm3. About 43(13.3%) and 25(7.7%) % had chronic comorbidities and opportunistic infection respectively. The majority of the participants were less than 10 years (53.1%) since using ART drugs. AZT-3TC-NVP (1c) is the most commonly used ART regimen at initiation (113(34.9%)), and the majority of the participants had regime change 305(94.2%) and (315(97.2%) use first-line regimen currently. Most participants 218(67.3%) use prophylaxis and within these prophylaxis users, INH is commonly used (134(41.4%)). , knee (20.7%), shoulder (10.6%), hip and thigh 9.5%, ankle and foot 8.9%, upper back 7.4%, hand and wrist 5.9%, elbow 4.5%, and neck 4%. . In addition, compared to those who used D4t-3TC-NVP (1a) type of ART, patients who was taking D4T-3TC-EFV (1b) was associated with an 81% reduction in the odds of developing MSDs , AZT-3TC-NVP (1c) had similarly an 81% reduction in the odds of developing MSDs  and AZT-3TC-EFV(1d) had 78% reduction in the odds of developing MSDs .Furthermore, Those HIV patients who didn’t change their first ART treatment regime had 8 times higher odds of developing musculoskeletal disorders compared to those who changed their first treatment regime [AOR = 8.14, 95% CI = 2.062 to 32.09. Patient with recent CD4 count 200–350 cells/mm3 had an 87% reduction in the odds of developing musculoskeletal disorders than patients with recent CD4 count > 350 cell/mm3.  , AZT-3TC-NVP (1c) , and AZT-3TC-EFV(1d)  were significantly associated with musculoskeletal disorders compared to D4t-3TC-NVP (1a) . This might be due to higher adverse effect of D4t-3TC-NVP (1a) like toxicity that leads to peripheral neuropathy which is characterized by pain and discomfort in any parts of their body  .Furthermore, HIV/AIDS patients who didn’t change their first ART regimen had higher odds of developing musculoskeletal disorders than those who changed their first ART regime. If ART initiated and remains in medication indefinitely there might be acute and chronic toxicities, concomitant clinical conditions, and development of virologic failure . Our stuThe prevalence of musculoskeletal disorder was moderate and the following factors are clinically significantly associated with musculoskeletal disorders among ART patient who are following at ART clinic: Recent CD4 count, opportunistic infection, ART regime at initiation and ART regime change. Thus HIV/AIDS patients who follow ART should be advised and educated to prevent opportunistic infection, use ART regime that have less impact in MSDs and to change their ART regime to prevent the long-term toxic effect of the drug. In addition, we recommend researchers to further investigate the effect of CD4 count for MSDs among HIV/AIDS patients following ART.This study assessed the burden of musculoskeletal disorder among HIV/AIDS patients following ART, which is the first study in Ethiopia. However, this study has certain limitations, the main limitation of this study was including small sample size. The cross-sectional design of the study precludes a follow-up, which would have provided a better design for detecting characteristics associated with musculoskeletal disorders. The results were also based on self-reported data from patients. This could have been influenced by recall bias.


Since January 2017, the recommended first-line antiretroviral regimen in Brazil is the fixed-dose combination of tenofovir plus lamivudine with dolutegravir (TL + D). According to the literature, integrase resistance-associated mutations (INRAMs) are rarely found upon virologic failure to first-line dolutegravir plus two nucleoside reverse transcriptase inhibitors. We evaluated the HIV antiretroviral genotypic resistance profile of patients referred for genotyping in the public health system who failed first-line TL + D after at least six months of therapy on or before December 31, 2018.HIV Sanger sequences of the pol gene were generated from plasma of patients with confirmed virologic failure to first-line TL + D in the Brazilian public health system before December 31, 2018.One hundred thirteen individuals were included in the analysis. Major INRAMs were detected in seven patients (6.19%), four with R263K, one with G118R, one with E138A, and one with G140R. Four patients with major INRAMs also had the K70E and M184V mutations in the RT gene. Sixteen (14.2%) additional individuals presented minor INRAMs, and five  patients had both major and minor INRAMS. Thirteen (11.5%) patients also presented mutations in the RT gene selected by tenofovir and lamivudine, including four with both the K70E and M184V mutations and four with only M184V. The integrase mutations L101I and T124A, which are in the in vitro pathway for integrase inhibitor resistance, were found in 48 and 19 patients, respectively. Mutations not related to TL + D, thus probable transmitted resistance mutations (TDR), were present in 28 patients (24.8%): 25 (22.1%) to nucleoside reverse transcriptase inhibitors, 19 (16.8%) to non-nucleoside reverse transcriptase inhibitors, and 6 (5.31%) to protease inhibitors.In marked contrast to previous reports, we report a relatively high frequency of INRAMs among selected patients failing first-line TL + D in the public health system in Brazil. Possible reasons for this discrepancy include delays in detecting virologic failure, patients inadvertently on dolutegravir monotherapy, TDR, and/or infecting subtype. Antiretroviral regimens containing integrase strand-transfer inhibitors (INSTIs) have been shown to have greater efficacy, safety, and fewer drug-drug interactions for the initial treatment of HIV infection than regimens containing non-nucleoside reverse transcriptase inhibitors (NNRTI) or protease inhibitors (PI) –6. AccorIn 1991, Brazil became the first middle-income country to provide free and universal access to antiretroviral therapy (ART) to all people living with HIV (PLWH) who qualify for treatment. Its Ministry of Health (MoH) periodically updates guidelines developed by an independent advisory panel. Since 2014, these guidelines have recommended that all PLWH be started on therapy regardless of symptoms or CD4 + T cell count .Starting in January 2017, the recommended first-line regimen in Brazil is the fixed-dose combination of generic tenofovir 300 mg plus lamivudine 300 mg, associated with 50 mg of the INSTI dolutegravir (DTG), a regimen known as TL + D. DTG is a second-generation INSTI with a high genetic barrier to resistance and few drug-drug interactions , 9. In aAlthough viral load monitoring is performed at the attending physician's discretion, measurements with intervals of less than six months are necessary for patients to continue to receive antiretrovirals through the Brazilian public health system .In 2001 the Brazil National AIDS Program created a national network for genotyping (RENAGENO), which developed methods and guidelines to standardize resistance testing in the public health system . Since JAccording to national guidelines, all patients who had at least six months of ART and confirmed virologic failure  are entitled to HIV genotypic resistance testing.As of December 2021, approximately 410,000 individuals were using DTG in Brazil, including those who had started DTG as first-line therapy, those who had switched to a DTG-containing regimen without having experienced prior virologic failures, or those who were using DTG after one or more virologic failures  . At the Here we present the results of 113 patients who failed the first-line TL + D regimen after at least six months of therapy and for whom resistance tests were requested to the national reference laboratory on or before December 31, 2018.pol gene were generated as previously described [This was a retrospective evaluation of genotypic resistance profiles of individuals with confirmed virologic failure after at least six months of first-line TL + D for whom resistance tests were requested to the Brazilian national reference laboratory before December 31, 2018. Confirmed virologic failure was defined as two successive detectable viral loads at least four weeks apart. Plasma samples from the second blood draw were evaluated. Sanger sequences of the integrase (IN), protease (PR), and reverse transcriptase (RT) regions of the escribed , 14. Resescribed . SubtypeThis study was approved by the local Ethical Review Board (# 19220719.7.0000.5505). The Brazilian MoH granted access to the national databases for PLWH . However, the Brazilian MoH had no role in the analysis or the interpretation of the data, and the views presented in this article are the entire responsibility of the authors.The Brazilian MoH has three large national databases for PLWH. The first, SISGENO, has the genotype results from all patients tested since the creation of RENAGENO in 2001, including FASTA sequence files, corresponding plasma HIV RNA viral loads, and CD4 + T cell counts results, all of which are automatically entered into the database. It also has information on treatment regimens being used by these patients.The second database, SISCEL, includes all lymphocyte CD4 + T cell and CD8 + T cell counts and viral loads performed in the Brazilian public health system since 1996. These data are automatically entered into the database through interfaces between the equipment performing the laboratory tests and the database.The third database is named SICLOM. The pharmacist responsible for each drug dispensation loads the data at the local dispensary. The software possesses several internal controls that prevent the distribution of drugs and/or regimens that are not in accordance with the national ART guidelines. For example, a patient being included for the first time in the system will only be allowed to receive TL + D. Special authorizations are necessary to dispense a different regimen, and justifications and names of the persons who authorized the dispensation are clearly marked in the system.To ensure that only patients failing first-line TL + D after six months of therapy were included in the present analysis, several precautions were taken. We first identified all first-line TL + D treatment failures in the RENAGENO database that occurred after July 2017. Next, we checked the SICLOM database to verify if each identified individual had not previously received antiretroviral drugs. In some cities, because data entry in the SICLOM database only became fully automated in 2011, we excluded patients from these locations from the analysis. If there was evidence of drastic declines in plasma viral load or significant CD4 + T cell count increases in the SISCEL database before initiation of TL + D, that patient was also excluded from the analysis as these changes might represent undocumented exposure to antiretroviral drugs.Data cleaning, organization, description, and statistical analysis were performed using the R Language and Environment for Statistical Computing  and its From July 2017 to December 2018, 113 antiretroviral naïve individuals who had started first-line TL + D had confirmed virologic failure after at least six months of treatment. Those who conformed to the inclusion criteria were identified and included in the present study. This series of patients includes all PLWH who failed first-line TL + D during the study period and for whom the attending physician requested a genotypic resistance test through the Brazilian public health system.10, whereas the mean CD4 + T cell count was 323.2. At virologic failure, the mean viral load was 5.13 log10, whereas the mean CD4 + T cell count was 365.9 . Among samples that failed to generate protease and reverse transcriptase PCR products, four were classified as subtype B, one as C, and one as F, according to the integrase profile. Of these, 77 (68.1%) were of subtype B, 12 (10.6%) of subtype C, three recombinant B/C , 12 (10.6%%) were of subtype F and three recombinant B/F . At the integrase gene, all 113 participants had subtypes recorded. Of these, 80 (70.8%) were of subtype B, 12 (10.6%) were of subtype C, four recombinant B/C (3.5%), 15 (13.3%) of subtype F, and two were recombinant B/F (1.8%).Integrase resistance-associated mutations were detected in 25 (22.1%) participants. Major INRAMs were detected in seven participants (6.19%): four with the DTG-specific mutation R263K, of whom three were clade B, and one clade C in the integrase gene. Of the other three participants with major INRAMs, one had the G118R mutation (clade C), one carried the E138A mutation (clade B), and one harbored the G140R mutation (BC recombinant virus). Two patients with the R263K INRAM also had both the K70E and M184V mutations in the RT gene.Eighteen (15.9%) additional individuals presented with minor INRAMs, 15 with a single mutation, and three with two mutations (M50I + L74I and M50I + G193E). It should be noted that five patients had both major and minor INRAMS, which were the association of R263K/R with M50I/T and L101I/V  participants presented with tenofovir and/or lamivudine resistance mutations in the RT gene, including four (3.5%) with both the K70E and M184V mutations. The other nine participants presented only the M184V mutation. One patient with the M184V mutation also had the thymidine analog mutation M41L mutation, which is not selected by either tenofovir or lamivudine.Drug resistance mutations (DRMs) not associated with resistance to tenofovir, lamivudine, or DTG, thus possibly transmitted drug resistance mutations, were detected in 28 participants 24.8%). These include 15 participants (13.3%) with DRMs to nucleoside reverse transcriptase inhibitors (NRTIs), 16 (14.2%) to NNRTIs, and 6 (5.3%) to PIs . The mean viral load among the seven individuals with major INRAMs was 5.17 log10 copies/mL. The difference between this value and those participants with no major INRAM is also not statistically significant .The mean viral load upon TL + D failure was 5.14 logp = 0.232). Table The mean CD4 + T cell count among individuals upon TL + D failure with no INRAMs was 383.06 compared to 287.56 among individuals with any INRAM. The mean CD4 + T cell count was 237.71 for the seven individuals with major INRAMs. These differences are not statistically significant . Other PLWH harboring HIV with this mutation showed viral loads of 4.6 log10 , and 5.6 log10 (patient 73). It has been shown that when drug-resistance mutations accumulate over time, there is a trend for viral fitness to be restored and for the viral load to increase [It has been demonstrated that specific DTG RAMs, such as the mutations at integrase codon 263, lead to strains with extremely low fitness  and low increase . It is pincrease  that we increase . Interesincrease , and a bincrease . Regardiincrease , 41.Of note, one patient infected by a recombinant B/C strain presented the recently described G140R mutation, associated G163R (Table In the present study, TDR was present in a quarter of the participants who failed first-line TL + D. This is significantly higher than previously reported in ART-naïve patients , 27, 45.In some of these cases, it is possible that TDR to other components of the regimen may play a role in the failures of first-line regimens containing DTG. Since many mutations associated with drug resistance may persist over time , pre-treIn Brazil, the only INSTI available before December 2017 was raltegravir, and its use from January 2009 to 2017 was limited to salvage therapy for patients with documented resistance to PIs. It was usually used in combination with one or more NRTIs, a boosted PI, with or without etravirine and/or maraviroc . AlthougThe prevalence of viremia above 50 copies/mL in clinical trials using DTG-based regimens for first line treatment is between 1 and 3% at weeks 24 or 48 . In 2017In marked contrast to what has been reported both in clinical trials and in clinical practice, we report a relatively high frequency of INRAMs among patients failing first-line therapy with a DTG associated regimen and two NRTIs in the public health system in Brazil. Possible reasons for this discrepancy include delays in requesting genotype testing, monotherapy with DTG by some patients due to selective inadequate adherence to the pill containing the NRTIs, TDR to other components of the regimen or to raltegravir, selection of mutations that are in the pathway to integrase inhibitor resistance, and the infecting subtype. Given their potential impact on global public health policies, these results merit further investigation.


Blips in HIV viral load (VL) while on antiretroviral therapy (ART) are of unclear clinical significance. In this study, we examined the association between blips and virologic failure (VF).We used data from the US Military HIV Natural History Study (NHS) cohort. Included participants were diagnosed with HIV after 2006, had been on ART for ≥ 6months, and had ≥3 VLs recorded and measured using an assay with a lower limit of detection of < 50 copies/mL. VF was defined as two consecutive VLs ≥ 200 copies/mL spanning 90 days or a single VL ≥1000 copies/mL. Blips were defined as VL of 51-999 copies/mL, that were preceded and followed by a VL ≤ 50 copies/mL and were differentiated by magnitude as low-level (VL 51-199 copies/mL) or high-level (200-999 copies/mL). Detectable VLs that did not meet criteria for VF or blips were grouped as low-level viremia , and higher low-level viremia . Cox proportional hazards models adjusted for demographic characteristics, time updated CD4 counts, ART, and viral load were used. Hazard ratios and 95% confidence intervals are presented.A total of 988 participants  were included, of which 55 (5.6%) experienced VF (Table 1). While 191 participants (19.3%) experienced blips, it was the highest VL status in 146 (14.8%) (Table 2). Having blips and low-level viremia was associated with increasing hazard of VF, graded by type and level of viremia  were protective (Table 3).Detectable viremia, including blips, are associated with an increased risk of VF. The magnitude of the viremia appears important with dose-response-type impact . These findings suggest that individuals with viral blips at a minimum should undergo evaluation for medication adherence and may benefit from more frequent VL monitoring.All Authors: No reported disclosures


The EuResist cohort was established in 2006 with the purpose of developing a clinical decision-support tool predicting the most effective antiretroviral therapy (ART) for persons living with HIV (PLWH), based on their clinical and virological data. Further to continuous extensive data collection from several European countries, the EuResist cohort later widened its activity to the more general area of antiretroviral treatment resistance with a focus on virus evolution. The EuResist cohort has retrospectively enrolled PLWH, both treatment-naïve and treatment-experienced, under clinical follow-up from 1998, in nine national cohorts across Europe and beyond, and this article is an overview of its achievement. A clinically oriented treatment-response prediction system was released and made available online in 2008. Clinical and virological data have been collected from more than one hundred thousand PLWH, allowing for a number of studies on the response to treatment, selection and spread of resistance-associated mutations and the circulation of viral subtypes. Drawing from its interdisciplinary vocation, EuResist will continue to investigate clinical response to antiretroviral treatment against HIV and monitor the development and circulation of HIV drug resistance in clinical settings, along with the development of novel drugs and the introduction of new treatment strategies. The support of artificial intelligence in these activities is essential. Although a cure for HIV cannot be achieved with available antiretrovirals (ARVs), the last 25 years saw dramatic improvement in the prognosis and management of HIV infection, thanks to the use of multiple drugs from different antiretroviral classes ,2,3. IndAs longer periods of follow-up data have become available, the clinician’s attention has shifted towards treatment tolerability and durability. Nevertheless, the impact of pre-existent drug-resistant mutations has remained a challenge in clinical practice, especially in PLWH exposed to suboptimal regimens in the first ART era . In addiIn 2006, a multidisciplinary group of scientists created the EuResist consortium to fight ARV resistance and obtained a grant by the European Commission under the 6th Framework Programme. The project integrated three existing national cohorts from Italy, Germany and Sweden to form a large dataset of clinical and virological information from around 17,000 PLWH, thus giving rise to the EuResist Integrated DataBase (EIDB). By modeling these data via statistical learning, the EuResist project delivered a clinical decision-support tool for predicting the best ART for specific PLWH by leveraging curated real-world data. The multicohort EuResist Network was established in 2008 as a follow-up of the EuResist project to manage the EIDB and the EuResist ART response prediction system. Currently, the EIDB is one of the world’s largest datasets suitable for training genotype-centered treatment-response models. The EIDB collects information about pseudonymized demographic and clinical characteristics of PLWH, including antiretroviral therapies, reasons for treatment change, AIDS defining events, viral co-infections, CD4 + T cells counts, viral load measurements, and HIV sequences.Originally, the EIDB was developed by integrating biomedical information from the three founding nationwide databases: ARCA , AREVIR (Germany), and InfCareHIV at Karolinska Institute, Stockholm (Sweden). Following the initial setup, other centers became partners of the EuResist Network and provided biomedical data to the EIDB. Most of these remain active contributors, together with the three founders; at time of writing, this includes: the Instituto de Higiene e Medicina Tropical ; the IrsiCaixa Foundation (Spain); the Laboratoire de Rétrovirologie of CRP-Santé (Luxembourg); the Gamaleya Institute of Virology (Russia); the Koacaeli University Medical Faculty (Turkey); and the CoRIS cohort (Spain). Additionally, data from the Rega Institute (Belgium); the National Referral Laboratory of Rwanda; the DUET study conducted by Tibotec; and the Resist study conducted by Boehringer Ingelheim, from the Infectious Diseases, AIDS and Clinical Immunology Research Centre of the State Institution Centre Public Health Ministry of Health of Ukraine and from the Infectious Diseases AIDS and Clinical Immunology Research Centre (IDACIRC) of Georgia were integrated in the past . https://www.euresist.org/eidb accessed on 31 March 2022). Data integrity and updating have been ensured for the last 15 years and onwards through an iterative process of data cleansing and periodical refresh from the contributing centers. The key uses of the EIDB have included the training of a machine-learning-based engine assisting treatment choices and providing curated datasets for specific studies, focusing on HIV variability, treatment, and drug resistance [The EIDB is updated twice a year with new data from the contributing centers by using import routines maintained centrally by the EuResist IT staff. Currently, it contains data from 105,903 PLWH, contributing 102,851 viral sequences, 248,249 treatment regimens, 1,451,753 viral load measurements and 1,486,295 CD4 cell counts A. The masistance .The objective of this manuscript is to offer an overview of the main contribution provided by the EuResist multicohort initiative in the fight against HIV drug resistance during the last 15 years, based on the efforts of many researchers from different European countries.The EuResist Network complies with the General Data Protection Regulation (GDPR) and all applicable laws, rules and regulations, with particular reference to ethical issues concerning the collection of data belonging to HIV patients and the relative manipulation and sharing procedures. All the data contributors to the EIDB certify that: the data collection was approved by the local ethics committees in agreement with local requirements and written informed consent was obtained from all patients before participation when necessary; the procedures are performed under the guidance and responsibility of the contributing centers; personal data are lawfully processed and may be contributed to the EIDB for scientific purposes on the basis of the data subject’s consent or another legal basis valid under the GDPR (art. 6 and art. 9).This study is performed in accordance with the ethical principles of the Declaration of Helsinki and the Good Clinical Practice guidelines of the International Conference on Harmonization.Data are securely stored in Italy. They contain sensitive human subject information and cannot be shared publicly because of privacy issues. Data are available under request to the EuResist Scientific Board, comprising all the data controllers. The EuResist Network has received research grants from the European Commission and unrestricted research grants from the following pharmaceutical companies: Abbott, Janssen Tibotec, Gilead Sciences, Merck Sharp and Dohme, Pfizer, Theratechnologies, and ViiV Healthcare. Neither the companies nor the patients had any role in the design, recruitment, or conduct of the studies.The emergence of antiretroviral drug resistance has long been a leading cause of treatment failure in PLWH. Particularly, before the advent of compact, high-genetic-barrier cART, the development of drug resistance was almost inevitable at some point in time. Thus, strategies for delaying the onset of resistance, preserving treatment options and avoiding cross-resistance were typically aided by algorithms interpreting HIV genotype. While these systems were mostly based on expert reviews of the literature, the EuResist consortium proposed a novel approach for predicting the in-vivo efficacy of antiretroviral drug regimens against a given virus isolate, based on statistical learning from a large amount of data correlating virus genotype and patient data on treatment response in clinical practice. vs human Experts) study in which 25 complete patient histories were provided to a panel of 10 global top experts in HIV drug resistance, who had to indicate whether the next ART regimen used would be effective or not. The EuResist system outcompeted nine of the experts in accuracy and equaled the most proficient one, demonstrating the strength and feasibility of the approach in a real-world simulation [Based on the analysis of the EIDB data, different predictive models have been developed over the years. In 2008, the first machine-learning engine was reported, which combined three models as follows: (a) generative-discriminative method, (b) regression with derived evolutionary features and (c) regression with a mixture of effects. The engine demonstrated an increase in the accuracy of prediction when taking into account clinical information and demographic factors in addition to genotypic information [mulation . In 2010, Bogojeska et al.  developehttps://hivdb.stanford.edu/ accessed on 10 April 2023) provides an impressive number of options for querying a curated HIV sequence database derived from published HIV sequences, with novel features and services released on a regular basis.It must be noted that, despite considerable efforts to improve the algorithms for prediction of response to ART, the increase in accuracy with respect to standard systems based only on the interpretation of HIV genotype has been limited. Factors impeding higher benefits by applying advanced statistics have not been identified but likely include the sparse representation of data in the training datasets and the complete lack of data on one major determinant of response to treatment, namely adherence to therapy. Moreover, the gain in accuracy comes at the cost of additional data to be input into the system—something that most doctors are reluctant to do. Additional bottlenecks for the transition from simple to improved yet more-complex methods include the lower degree of human interpretability of the latter and the globally decreased impact of drug resistance in modern ART . As a maThanks to the continuous and longitudinal accumulation of viral sequences and patient data, the EIDB has offered a tremendous opportunity to monitor the selection and spread of transmitted and acquired resistance-associated mutations (RAMs), the circulation of different viral subtypes, and their impact on treatment outcome, most often as measured by changes in viral load. In more recent years, non-B subtypes have become endemic in the European population, likely due to human migration from non-B areas. This may have had an impact on the global picture of drug resistance since most non-B areas have been characterized by a different coverage of ART regimens and consequently different patterns of emergent drug resistance. This effect has been later diluted and will continue to be diluted by the harmonization of ART regimens, particularly those based on high-genetic-barrier regimens, significantly limiting drug resistance and failure.A key issue in HIV drug resistance is the prevalence and incidence of multidrug resistance, resulting in an extensive loss of treatment options and the inability to control virus replication and halt disease progression. Two EuResist studies have shown a declining trend of resistance to the four main classes of ARVs, including protease inhibitors (PI), nucleoside (NRTI) and non-nucleoside (NNRTI) reverse transcriptase inhibitors, and integrase strand transfer inhibitors (INSTI). The first study analyzed viral sequences collected from 3414 individuals with at least 1 genotype test result available between 2008 and 2019 and found that high-level resistance to at least 1 drug in each of the 4 main ARV classes declined from 5.6% in 2008 to 2.4% in 2018, with only 2.5% of the patients accumulating four-class resistance over time . NotablyThe studies conducted by Lapokov et al.  and NeshA recent study conducted by van de Klundert et al.  showed, Over the years, the EIDB was used to explore the clinical impact of specific RAMs. For example, Theys et al.  observedInspired by the updates of the international HIV treatment guidelines, the EuResist Network explored the real-world effectiveness of different treatment strategies. Understanding and predicting treatment efficacy and duration of effectiveness, i.e., how long an antiretroviral therapy can be sustained without changes, even in the presence of pre-existing RAMs, has become an important challenge in the context of addressing HIV treatment optimization. For example, the analysis of the EIDB data suggested that the efficacy of the innovative dual-drug strategy combining the NNRTI etravirine with the ritonavir-boosted PI darunavir was comparable to that of alternative regimens where etravirine was coupled with an alternative boosted PI . In 2019More recently, two studies explored the effectiveness and discontinuation rate of INSTI, a key component of most current ART regimens. Among 2976 INSTI treatments started by naïve PLWH, elvitegravir- or dolutegravir-based first-line antiretroviral treatment was highly effective in routine practice across Europe, even in the presence of transmitted drug resistance for the accompanying NRTIs. However, the rate of INSTI discontinuation was much higher than the rate of virological failure . InteresAnother study by Miranda et al.  investigThe EuResist Network has been collaborating with other non-European cohorts and global initiatives to investigate HIV-resistance-related issues, particularly in low-income countries. From 2010 to 2013 it participated in the ENEAA project (EDCTP JP.2009.10800.002), coordinated by Karolinska Institut and involving the Addis Ababa University, Ethiopia; the Muhumbili hospital, Dar-es-Salaam, Tanzania; and the World Friends Hospital, Nairobi, Kenya, integrating training activities and IT infrastructures to improve capacities in the eastern African area. The TenoRes collaboration included data from adult HIV treatment cohorts and clinical trials in Europe, Latin and North America, sub-Saharan Africa, and Asia. Among 1926 PLWH enrolled after virological failure with first-line tenofovir-containing ART, HIV drug resistance was detected in an alarmingly high fraction of cases, strengthening the need for the surveillance of transmitted drug resistance . AnotherThe EIDB can be accessed upon formal request by scientists aiming at investigating HIV treatment and drug-resistance-related topics. The EuResist Management Board processes requests in a reasonable time and asks the individual cohorts for permission to access the data for the specific study, which has to be well-defined and scientifically sound. EuResist encourages other clinicians and scientists, also from non-European countries, to collaborate for the advancement of knowledge on HIV drug resistance and treatment, and it is open for scientific studies. Karolinska Institutet  ;Max Planck Gesellschaft (Germany);University of Siena ;InformaPRO s.r.l. ;Cologne University (Germany).EuResist Network is a non-profit European Economic Interest Grouping (EEIG) composed by:th Framework Programme (FP)), the EuResist Network participated in and guided several research projects funded by the European Commission, among which we can mention: CHAIN-Collaborative HIV and Anti-HIV drug resistance Network, a large scale integrated project ; CARE-Common Action against HIV-TB HCV across the Regions of Europe, ; and EuCARE-European Cohorts of Patients and Schools to Advance Response to Epidemics , where the focus moves to SARS-CoV-2 ad its variants.In addition to the already mentioned EuResist project (Grant n. IST-2004-027173, 6EuResist also runs dissemination and training activities. Further to the cited ENEAA EDCTP project, it offers training tools on its website; it participates in the annual ARCA Mentor School event, and has organized a EuResist Mentor School in Cologne in 2018 with the objective to bridge the gap between data science and medical/biological sciences.Since 2007, EuResist has contributed to the annual Arevir Meeting organized by the University of Cologne and Genafor, holding a EuResist session.The main strength of the EuResist Network is the inclusion of all PLWH followed at each of the participating centers, ensuring the full representativeness of the real-life setting, including individuals typically not enrolled in randomized trials due to, e.g., the presence of baseline drug resistance or past virological failures. The sample size of the cohort is also notable, with a total of 105,903 PLWH as of March 2022. Likewise, the geographic distribution of the participating centers is Europe-wide, with additional cases from Turkey, Russia, Ukraine, Georgia, and Rwanda. The observational, retrospective, and prospective multicohort design provides an extraordinarily large time span, with data collection starting as early as the late 1980s, translating into a unique opportunity to follow virus evolution and investigate very different treatment strategies across all antiretroviral treatment eras.The main limitation of the EIDB is that most contributing cohorts collect only routine clinical and viro-immunological data. Some data can be missing in the individual databases, making the overall data collection not uniform across areas and resulting in the exclusion of large datasets when specific variables are required. The lack of information on tolerability issues, adherence to ART, clinical non-AIDS-related events and concomitant medications make the cohort not suitable for many kinds of investigations. Lastly, although a semiannual update is planned, delays between local data collection and the availability of an updated centralized database often occur, postponing the possibility to study the most recent treatments, which are typically of maximum interest.The EuResist cohort was established with the purpose of developing a clinical decision-support tool predicting the most effective antiretroviral therapy for PLWH based on their clinical and virological data. Later, the cohort widened its activity to the more general area of antiretroviral treatment with a focus on virus evolution and drug resistance. Following its interdisciplinary vocation, EuResist will continue to investigate clinical response to antiretroviral treatment against HIV and monitor the development and circulation of HIV drug resistance in the clinical setting along with the development of novel drugs and the introduction of new treatment strategies. The support of artificial intelligence in these activities is essential.


Compared with baseline mean CD4 + T-cell counts of 122.72 cells/μL, it increased to 207.87 cells/μL (P = .0067) and 218.69 cells/μL (P = .0812) after 4 and 8 weeks of treatment. Except for limited laboratory abnormalities such as hyperuricemia, increased creatinine level, and hyperglycemia observed after treatment, no other clinical adverse events were considered related to ABT plus DTG. Data suggests that ABT plus DTG is safe and effective for critically-ill hospitalized PLWHA. In view of the rapid viral load suppression and restoration of CD4 + count within 8 weeks of treatment, its clinical application warrants further investigation.Treatment options for hospitalized people living with HIV/AIDS (PLWHA) with opportunistic infections and comorbidities are limited in China. Albuvirtide (ABT), a new peptide drug, is a long-acting HIV fusion inhibitor with limited drug-drug interactions and fast onset time. This single-center, retrospective cohort study investigated the effectiveness and safety of ABT plus dolutegravir (DTG) therapy in a real-world setting. We performed a chart review on the electronic patient records for hospitalized PLWHA using ABT plus DTG between April and December 2020. The clinical outcomes were retrospectively analyzed. Among 151 PLWHA (mean age 47.6 ± 15.9 years), 140 (93%) had at least 1 episode of bacterial and/or fungal infections and 64 (42%) had other comorbidities including syphilis, hepatitis B, and/or hypertension. ABT plus DTG was given to 87 treatment-naïve (TN) and 64 treatment-experienced (TE) PLWHA. Regardless of treatment history, mean HIV-1 RNA levels significantly decreased from 4.32 log Among people living with HIV/AIDS, wasting, the onset of opportunistic infections, chronic comorbidities, and drug toxicities can be causes for hospitalization.Treatment options are limited for hospitalized people living with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) with opportunistic infections and comorbidities. With high HIV-RNA levels and low CD4 + T-cell counts, people living with HIV/AIDS are at high-risk of advanced infections, However, there is a lack of robust evidence to support ART regimens use in individuals with critical-ill HIV disease. Critical-ill HIV-infected patients are admitted to the hospital for AIDS-related events or organ dysfunctions with critical care management. The decision to initiate or maintain highly active antiretroviral therapy in this situation is subjective, as there is no current consensus among experts. The ART choice for critically-ill populations with advanced HIV infection/AIDS are limited due to drug-drug interactions, overlapping toxicities, high pill burden and the possibility of immune reconstitution inflammatory syndrome.–10 In China, efforts have been made to increase the coverage of ART for people living with HIV/AIDS.–14 Nevertheless, poor treatment compliance and ART drug intolerance remained a concern and subsequently increasing treatment failure, emergence of drug resistance, suggesting an imminent need for alternative therapy.Anti-retroviral therapy (ART) has been proven effective in reducing viral load (VL), virulence, as well as morbidity and mortality of AIDS.–18 ABT was proven to exhibit a broad spectrum of anti-HIV-1 activity, including viral strains predominantly circulating in China and some variants resistant to enfuvirtide (T20). In a prospective Phase II clinical study of anti-retroviral therapy (ART) – naïve subjects, the 2-drug regimen ABT plus ritonavir-boosted lopinavir (LPV/r) was associated with significant decreases in HIV-1 viral load. Through the Phase III TALENT study, ABT plus LPV/r was found non-inferior to standard WHO-recommended second-line LPV/r with 2NRTIs. Since ABT becomes available in China, real-world experience showed that it is well-tolerated with limited treatment-emergent adverse events and expanded uses were found especially for people who are of higher unmet needs and running out of treatment choices. Considering ART to combine with ABT, LPV/r may have several limitations including adverse events such as gastrointestinal intolerance and elevated lipid levels and potential drug-drug interactions with anti-fungal drugs, anti-tuberculosis drugs etc. DTG is now a first-line medication worldwide due to its high genetic barrier to resistance and superior efficacy. Another trial studied 28-days ABT plus dolutegravir (DTG) versus oral drug combinations of DTG, tenofovir disoproxil fumarate plus lamivudine (3TC) as post-exposure prophylaxis, and found the combination to be well-tolerated and effective with significantly greater adherence and completion rate than the oral drug regimen. Therefore, it is of interest to explore DTG, with the newly approved long-acting injectable fusion inhibitor for advanced, hospitalized HIV-1 infected patients. The present retrospective study aimed to investigate the clinical outcomes and safety of ABT plus DTG in a real-world setting.In this context, the dual regimen contains albuvirtide (ABT) plus dolutegravir (DTG). ABT is a chemically modified peptide that targets the HIV-1 gp41 envelope protein and inhibits the formation of the gp41 6-helix bundle structure (6-HB), which is necessary for the fusion of the viral and cellular membranes. ABT is a long-acting, once-weekly injectable HIV fusion inhibitor with few drug-drug interactions and a half-life of 11 to 12 days.A retrospective cohort study was conducted using electronic patient records from Chengdu Public Health Clinical Medical Center. This study was approved by the Ethics Committee of Chengdu Public Health Clinical Medical Center  and the requirement for written consent was waived due to this study used routinely collected clinical data in an anonymized format.We reviewed ART records for hospitalized people living with HIV/AIDS (PLWHA) from April to December 2020 and included all patients at site who received ABT combined with DTG during hospitalization. Records with the following criteria were included in the analysis: people living with HIV/AIDS with opportunistic infections and/or exacerbation of comorbidities who were in critical condition; aged more than 18 years old; started antiviral treatment involving DTG 50mg once daily or twice daily and ABT 320mg IV once daily on day 1, 2, 3, and day 8 weekly onwards. Records were excluded for patients who were not tested for the plasma HIV-1 RNA and CD4 + T lymphocyte count after administration of ABT. A flowchart detailing the patient evaluation process was shown in Figure  Hospital discharge data was recorded and available, no long-term survival data was recorded in this study.Clinical records of plasma HIV-1 RNA measured via Real-Time polymerase chain reaction and peripheral blood CD4-cell counts measured using a FACS count system before treatment, 2 weeks, 4 weeks and 8 weeks after treatment were retrospectively reviewed and analyzed. Toxicities were reviewed from the medical and laboratory records and were graded and evaluated according to Common Terminology Criteria for Adverse Events 5.0.P value of the change from baseline were reported by each visit, as well as P value of the average change from baseline of postbaseline versus baseline comparison. Log10 transformed data was applied in MMRM for HIV analysis, original data was applied in MMRM for CD4 and CD4/CD8 analysis. 95%CI for each visit was calculated based on Clopper-Pearson method, and 95%CI for the difference was calculated based on Miettinen and Nurminen (MN) method in analyzes for HIV RNA suppression in subgroup. The differences of viral load suppression rates between subgroups were analyzed by Chi-square tests. The results were considered statistically significant at P < .05. We used SAS version 9.4  for all statistical analyses.The baseline characteristics of patients were presented by descriptive analysis. Numerical data with a normal distribution are presented as mean and standard deviations (SDs) and those with non-normal distribution are summarized as medians and interquartile ranges. Analyses on the changes of HIV-1 RNA, CD4-cell counts and the ratio of CD4/CD8 along the treatment periods were performed using a mixed-effects model for repeated measures (MMRM) without imputation. Kenward-Rogers approach was used for degrees of freedom approximation. The Least Squares Means, 95% CI and Between April and December 2020, 151 critically-ill, hospitalized people living with HIV/AIDs treated with ABT plus DTG were retrospectively evaluated for the anti-viral effectiveness and treatment safety in clinical settings. Patients ranged from 21 to 87 (mean: 47.6 ± 15.9) years with over 55% of cases younger than 50 years old, and more than 80% being male. Among all patients in this study, 140 (93%) had at least 1 episode of bacterial and/or fungal infection and 64 (42%) had other comorbidities such as syphilis, hepatitis B, hypertension etc. A total of 87 (57.6%) patients were treatment-naïve (TN) and 64 (42.4%) were treatment-experienced (TE). Baseline characteristics are shown in Table Most patients had comorbidities or concomitant treatments Table . ABT plu10copies/mL to 2.24 log10copies/mL, 2.10 log10copies/mL and 1.89 log10copies/mL after 2, 4 and 8 weeks of treatment, respectively (P < .0001) (Table P = .0067) but insignificantly to 218.69 cells/μL after 8 weeks of treatment (P = .0812) when compared to baseline. Further analysis revealed a significant increase of the mean CD4 + T-cell counts in TN patients from 90.48 cells/μL to 182.49 cells/μL and 222.99 cells/μL after 4 and 8 weeks of treatment (P < .0001), respectively. However, the increment of mean CD4 + T-cell counts in TE patients was significant from a baseline of 166.55 cells/μL to 221.60 cells/μL after 4 weeks of treatment (P = .0117) but insignificant to 203.79 cells/μL after 8 weeks of treatment (P = .1249). The mean CD4/CD8 ratio increased from a baseline of 0.28 to 0.31 after 4 weeks (P = .1617) and significantly to 0.38 after 8 weeks (P = .0008) of treatment in all patients. A statistically significant increase in the ratio was observed after both 4 and 8 weeks of treatment among TN patients and 4 weeks of treatment among TE patients.Regardless of treatment history, mean HIV-1 RNA levels significantly decreased from 4.32 log10copies/mL to 1.88 log10copies/mL (P < .0001), as well as significant increases in CD4 + T-cell from 140.4 cells/μL to 259.9 cells/μL (P = .0025), and increasing CD4/CD8 ratio from 0.34 to 0.47 (P = .0431) after 8 weeks of treatment (Table P = .2118) (Table P = .0912) (Table 10copies/mL to 1.89 log10copies/mL (P < .0001), while mean CD4 + T-cell count was increased significantly from 97.86 cells/μL to 221.52 cells/μL (P < .0001) among the group of patients with detectable baseline VL  and TE patients (92.2%) achieved viral suppression after 8 weeks of treatment. ABT plus DTG was highly effective in TE patients either with undetectable baseline viral load or virological failure. The results are consistent with the TALENT study, 75.7% of study participants treated with ABT/LPV/r achieved HIV-1 RNA levels below 40 copies/mL after 48 weeks of treatment. Highly active antiretroviral therapy can induce sustained recovery of CD4 + T cell reactivity against opportunistic pathogen in severely immunosuppressed patients. Decreasing viral load can reverse HIV-driven activation and CD4 + T cell defects in advanced HIV-infected patients. While promising effectiveness was observed in this retrospective study, a few potential prognostic predictors were examined. A low CD4/CD8 ratio was considered predictive of poor prognosis with non-AIDS related events or death even during effective ART.,38 In our study, the CD4/CD8 ratio did not increase significantly among participants previously treated with ART, although the ratio slightly improved after 8 weeks of treatment, which might reflect lower immunological recovery for TE individuals than TN people living with HIV/AIDS. As this study did not provide data on optimal treatment duration beyond 8 weeks, additional research is needed to determine the disease prognosis after a longer treatment duration.Another key finding is the significant immune recovery after 8-week treatment of ABT + DTG. The result is more prominent in TN patients. The mean CD4 + T-cell counts increased by > 100 cells/uL after 8 weeks of treatment from baseline. Another study by Yang J. et al also reported an increased trend of CD4 + cell count in treatment naïve patients using ABT monotherapy for 30 days. Good tolerability and low drug-drug interactions are critical considerations for treatment selection.In our study, ABT plus DTG were well tolerated in this group of critical-ill patients living with HIV/AIDS, reflected by no adverse reactions related to the study drug and no ABT-emergent discontinuation cases. WHO recommends patients with advanced HIV should have rapid ART initiation .This retrospective cohort study investigated the use of ABT plus DTG dual therapy in a real-world setting. The treatment outcome and safety profile should be carefully interpreted and several limitations should be noted. This study does not answer the question on the efficacy and safety of ABT plus DTG versus standard-of-care treatments because this was a single-arm study. Data of patients was only recorded up to 8 weeks or shorter and for routine clinical care without long-term follow-up. There is a need for a well-controlled study to investigate which populations may derive the optimal benefit of ABT-based therapy, as well as the optimal treatment duration for ABT plus DTG to achieve and prolong therapeutic outcomes. An extended review on the survival outcomes of patients treated with ABT may help determine the long-term treatment impact. Such investigations could also include the efficacy of ABT in individuals with T20 peptide resistance.This retrospective cohort study showed that ABT plus DTG therapy in clinical practice provided a high rate of viral suppression and restoration of CD4 + T cell count within 8 weeks of treatment. No significant safety concerns were observed. Therefore, in the short term, the ABT-based regimen is well tolerated, and the viral load quickly becomes undetectable, but it remains to be investigated if this is maintained over time.The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Health Planning Committee Project Foundation of Sichuan [Grant numbers 18PJ341].Investigation: Huanxia Liu, Shenghua He, Tongtong Yang, Chunrong Lu, Yuan Yao, Ruifeng Zhou, Ke Yin, Yuanhong He, Jing Cheng.Methodology: Huanxia Liu.Project administration: Huanxia Liu.Supervision: Shenghua He.Writing – original draft: Huanxia Liu.Writing – review & editing: Shenghua He.


The cumulative frequency of all cause cART modification was 3223 (64%): 2956 (58.8%) substitutions; 37 (0.7%) switches; and both, 230 (4.5%). Following 241,194 person-months (PMFU) of follow-up, incidence rate of cART substitution and switch were 12.3 (95% CI 11.9–12.8) per 1000 PMFU and 3.9 (95% CI 3.2–4.8) per 10,000 PMFU, respectively. Prominent reasons for cART substitution included toxicity/intolerance, drug-shortage, new drug availability, treatment failure, tuberculosis and pregnancy. The most common adverse event (AEs) associated with cART modification included lipodystrophy, anemia and peripheral neuropathy, among others. In the adjusted multivariate Cox regression model, Organisation  ; Initial WHO clinical stage ; NRTI backbone  were associated with increased cumulative hazard of treatment modification. Baseline weight ; address within Maekel ; AZT-based backbones ; TDF-based backbones: aHR = 0.068, 95% CI 0.051–0.091, p value < 0.001), NVP-based anchors  were associated with lower cumulative hazards of attrition. The minimal number of switching suggests inadequate VL testing. However, the large number of toxicity/intolerance and drug-shortage driven substitutions highlight important problems in this setting. Consequently, the need to advocate for both sustainable access to safer ARVs in SSA and improvements in local supply chains is warranted.Combined antiretroviral therapy (cART) durability and time to modification are important quality indicators in HIV/AIDs treatment programs. This analysis describes the incidence, patterns, and factors associated with cART modifications in HIV patients enrolled in four treatment centers in Asmara, Eritrea from 2005 to 2021. Retrospective cohort study combining data from 5020  patients were utilized. Data on multiple demographic and clinical variables were abstracted from patient’s charts and cART program registry. Independent predictors of modification and time to specified events were evaluated using a multi-variable Cox-proportional hazards model and Kaplan–Meier analysis. The median (±IQR) age, CD4 Fundamentally, the report suggests that more than half the global population of people living with HIV/AIDS (PLWHA) are found in ESA and that the region has the highest incidence of AIDS-related mortality, 310,000 . Fortunately, the report notes that combination antiretroviral therapy (cART) coverage in the region has greatly improved and that currently 16.0 million [15.4 million–16.1 million] or 77% [60–92%] are accessing treatment1.The 2021 Joint United Nations Program on HIV/AIDS (UNAIDS) fact sheet estimated that in 2020, there were 37.7 million [30.2 million–45.1 million] people living with Human Immunodeficiency Virus (HIV) including 20.6 million [16.8 million–24.4 million] in Eastern and Southern Africa (ESA)2. Despite these positives and the decade-long successes in improvements of cART coverage in Low and Middle Income Countries (LMICs); treatment programs in the region are still facing multiple challenges. These include limited access to experts and relevant diagnostics , clinical chemistry, and genotype testing). More importantly, the use of second-generation Non-Nucleoside inhibitors (NNRTIs), Integrase inhibitors [INSTIs], fixed-dose cART formulations without older Nucleoside Reverse Transcriptase Inhibitor (NRTI) backbones, or newer, less toxic drug classes like Entry inhibitors (EIs) are still not part of the standard of care. Instead, cART availability in most Sub-Saharan Africa (SSA) countries is restricted to a limited formulary comprising of a small-number of well-studied combination of old anti-retroviral (ARVs). This development is largely attributable to the World Health Organization (WHO) proposition that countries in the region should adopt a public health based approach to cART [WHO 2006]3.The benefits associated with improvements in cART are adequately described in existing literature and these include sustained virus suppression, immune reconstitution/recovery, and dramatic reductions in HIV/AIDS morbidity and mortality. Low viremia is also associated with reduced HIV transmission rates5. At present, the most straight forward pathway to maximization of cART durability involves the adoption of prudent modification strategies—decreasing substitution rates and limiting unnecessary switching to second-line regimens. Multiple studies have confirmed drug modifications in cART care settings can be prompted by a number of medication and patients-dependent factors. The former includes the need to limit adverse events (AEs); treatment failure; new drugs, eliminate drug—drug interactions (DDIs); reduce pill burden or dosing frequency; and availability of new drugs. On the other hand, there are many well-recognized concerns regarding inappropriate modification of cART. Possible risks include the development of viral cross-resistance to specific ARVs6 and increased mortality7. This may compromise the preservation of these medications as components of alternative first-line cART regimens and/or second line options8. Importantly, data suggests that subsequent regimens have progressively shorter durability and are costlie9. These, it has been suggested; are important concerns in SSA where cART options are severely limited6.Due to the limited number of options, particularly anchor drugs; maximization of first or second line regimen durability is recognized as a key programmatic priorityAltogether, we can argue that despite the necessary occurrence of treatment modifications in the lifelong course of cART; frequency, patterns and correlates of treatment modifications (particularly in the pre- and post-INSTI era) are poorly characterized in SSA. Therefore, the primary objective of this study was to describe incidence, patterns, and factors associated with drug modifications in patients on cART in multiple treatment centers in Asmara, Eritrea. This information can be leveraged to develop preventive interventions aimed at enhancing durability of the first regimen.10. Treatment eligibility has evolved tandem with WHO recommendations—CD4+ T-cell count of ≤ 200 cells/μL; ≤ 350 cells/μL; ≤ 500 cells/μL, and test-and-treat approach in 2005, 2010, 2015, and 2016, respectively.We conducted a retrospective cohort study at four cART centers in Asmara, Eritrea. These hospitals include: Orotta National Referral Hospital (ONRH); Halibet National Referral hospital (HRH); Sembel hospital (SH); and Haz-Haz Zonal Referral Hospital (HzH) located in Asmara, the capital city of Eritrea. The central zone is the largest administrative region out of the six zones in the country. Before the decentralization of services to other zones in 2010; these were the only institutions that were offering ART to people living with HIV in the country. In total, around 6235 adults have accessed cART in these clinics since the inception of the program in 2005. Treatment is guided by the Eritrean National Adult guidelines developed by Eritrean Ministry of Health (EMoH)gratis. Dispensing of cART occurs after every three months or as per scheduled clinical visits. During these visits, additional care , among others) is provided.Drug combinations are designed as per WHO recommendations around the following ARVs: Nucleotide reverse transcriptase inhibitors (NRTI) (Zidovudine (ZDV), Lamivudine (3TC), Emtricitabine (FTC), Tenofovir disoproxil fumarate (TDF), Didanosine (DDI), Abacavir (ABC)—Stavudine (d4T); Non-nucleoside reverse transcriptase inhibitors (NNRTIs): Nevirapine (NVP), Efavirenz (EFV); Protease inhibitors (PIs): Lopinavir/ritonavir (LPV/r); and Intergrase inhibitors (INSTIs) (Dolutegravir (DTG). Major guidelines changes were adopted in 2010. Integrase Inhibitors (DTG in particular) was not readily available before 2020. These drugs along with required laboratory services are provided All adult patients with HIV/AIDS who attended the four outpatient HIV follow-up clinics from 2005–2021 were enrolled in the study. All patients included in the analysis were initiated on a variety of cART backbones . Anchor drugs include ATV/r, DTG, EFV, LPV/r, and NVP. Excluded individuals were: those whose records were repeated in another hospital, incomplete data of cART regimen modification and/or illegible. As per the standard care at the HIV clinics, all the patients had their HIV-1 infection status confirmed positive by nucleic acid based testing  TaqMan1 HIV-1 Test). See Fig. + T-cell count cells/mm3). Regimen data assessed included First-line cART combination, subsequent modifications (substitutions and/or switching) and reasons for cART modifications. The process was undertaken by trained health professionals and was closely monitored by the principal investigator and supervisors. Data collection was carried out in September-December 2021.De-identified and anonymous patient data were obtained from the cART registries of the respective hospitals and patient cards. Accordingly, all clinical cards were reviewed for demographic information ; clinical , and laboratory data  for class (NRTI or NNRTI) replacement of one or more First-line cART regimen6. Due to the fact that 3TC and FTC are therapeutically interchangeable, a change between the two drugs was not regarded as a substitution.Drug Switching: Drug switch was defined as a change from the first-line to Second line regimen.The primary outcome of this study was to determine the factors associated with cART modification. This was evaluated between the first documented time of cART initiation (baseline) and the last time occurring within the end of the study period . In the process, the following operational definitions were used:2)/Fishers exact test and summarized using counts (frequency) and proportions (percentages). Normality test was conducted prior to any statistical computation and quantitative data was summarized using median with Interquartile range (IQR). The incidence rate of drug modification was calculated by dividing the number of patients with initial first-line ART modification by the total number of persons-years of follow up. Furthermore, Kaplan–Meier estimates and log-rank tests were carried out to compare the cumulative incidence of cART modification between different categories of patient specific characteristics. Finally, a Cox regression hazard was performed to identify the predictors of therapy modification. The variables that were considered in a multivariate-adjusted Cox proportional hazards model of drug modification were age at treatment initiation, gender, baseline functional status, baseline clinical status, baseline CD4+ count and baseline body weight. A backward-selection procedure was used to create these adjusted models, with a variable being included in the model if it resulted in an improvement in the model fit. The final results are presented as AHR with a 95% confidence interval (CI). In all instances, p value < 0.05 was regarded as significant.Analysis was conducted using IBM SPSS version 26  and Stata version 14.0 . Descriptive statistics for categorical variables were analyzed using chi-square  research ethical committee (Letter of reference: # 08/2021) and communicable disease control (CDC). All the information gathered was De-identified, held with utmost confidential responsibility and used only for this study’s purpose.As the study also included data based on patients’ clinical card records, consent for the data access was waived by the ethical committee in place of the patients. This study conforms to the principles outlined in the Declaration of Helsinki.Ethical clearance was obtained from the Health Research Ethics and Protocol Review Committee of the Ministry of Health. Consent to participate was not obtained from the patients. All ethical and professional consideration were followed during the study to make patients identity strictly confidential.+ cell count was 160 (IQR 80–271) cells/µL with a significant proportion of participants, 3083 (61.3%), having CD4+ T-count < 200 cells/mm3. See Fig. A total of 5020 patients  with 5.8 (IQR 3.4–9.4) years of follow-up time enrolled in the study. The median (± IQR) age at cART initiation was 48 (IQR 41–55) years and participation favored females, 3077 (61.3%). Majority of participants were from central zone 4219 (84%), married 2262 (45.1%) and had secondary level education 2109 (42%) while nearly half of patients were employed 2221 (44.2%). In terms of disease clinical conditions, 2667 (53.25%) were either in WHO stage III or IV. Moreover, the median (± IQR) baseline CD49.5%, HosPrior to the WHO consolidated guidelines updates of 2010, the most frequently prescribed initial regimen was d4T/3TC/NVP or EFV 35.1%) and AZT/3TC/NVP or EFV (35%) , 31 (83.8%) and 206 (89.6%), p value = 0.002. Further, median (± IQR) CD4+ T-cell count was associated with substitution, switching or both—158.5 cells/mm3 (82–248 cells/mm3), 113 cells/mm3 (66–309 cells/mm3), 140 cells/mm3 (65–213.5 cells/mm3), Kruskal–Wallis test p value < 0.001. Concerning cART regimen, ABC/3TC, AZT/3TC, D4T/3TC were associated with higher proportions of substitution (80 (76.9%) vs. 1312 (74.7%) vs 1472 (83.6%), respectively) compared to TDF/FTC based back bones (92 (6.6%). Similarly, EFV was associated with lower substitution, 887 (30%) vs. 2068 (69.9%) for NVP. Additional factors that were associated with substitution, switching or both included occupation, marital status, baseline weight in Kg, baseline WHO clinical stage, and baseline functional status. See Table Of the 2956  patients who incurred a drug substitution at least once, 1560 (52.5%) involved a single NRTI substitution, 310 (10.43%) involved a single NNRTI substitution, and 1075 (36.2%) involved both an NRTI and an NNRTI substitution. Further, 230  patients experienced both types of cART modification (drug substitution and drug switch) while 37  patients switched from NNRTI-based to PI-based regimen. Hospital C had significantly higher proportion of drug substitution 1090 (36.9%) and switching 15 (40.5%) or both 94 (40.9%) patients. High record of cART switching was associated with lower median age ; st-5th modification events were lipodystrophy, 481 (27.36%); anemia, 144 (8.19%); peripheral neuropathy, 96 (5.46%) among others. In the NRTIs group, adverse events observed included lipodystrophy, 481 (27.35%); Anemia, 144 (8.19%); peripheral neuropathy, 96 (5.46%), and skin rash, 72 (4.09%).Figure th substitutions.The most prescribed cART backbones at the start of the programme in 2005 were d4T/3TC, 169 (59.1%), and AZT/3TC, 103 (36%). The dominant anchor drug in the same period was NVP, 251 (87.8%). Treatment initiations on d4T/3TC cART backbone persisted until 2011 and dropped drastically, in 2012 to 30 (6.36%). In 2011, the dominant cART backbone was AZT/3TC, 232 (52.6%). Treatment initiation on TDF/FTC cART increased from a low point of 48 (10.9%) in 2011 to a peak of 113 (89.7%) before declining to 41 (50.3%) in 2021. The use of EFV as an anchor drug at cART initiation increased gradually from 35 (12.2%) in 2005 to 45 (88.2%) in 2019. DTG usage was initiated in 2020. See Fig. p value < 0.001]. Moreover; patients aged > 60 years were observed to have significantly shorter mean duration to substitution . WHO clinical stage 3 and 4, CD4+ T-cell count < 200 cells/µL and bed ridden patients were also observed to have shorter mean duration to drug substitution. Regarding ART regimen, TDF/FTC backbone and NVP from the NNRTI showed significantly longer mean duration to substitution. Concerning cART switching, patients followed in Hospital A had longer mean duration to ART switch of 185 (95% CI 183–187) months as compared to Hospital B  months, Hospital C  months and Hospital D  months; log rank p value = 0.002. See Table Following 241,194 person-months of follow-up (PMFU), incidence rate of cART substitution and switch was 12.3 (95% CI 11.9–12.8) per 1000 PMFU and 3.9 (95% CI 3.2–4.8) per 10,000 PMFU, respectively. Kaplan–Meier survival analysis for cART substitution and switch was then conducted to compare the mean duration of survival among sub-groups of patient characteristics. In this analysis, patients followed in Hospital A had significantly shorter mean duration to drug substitution  PY observation+ T-cell count and clinical presentations. In the absence of routine HIV RNA-VL monitoring, switching occurred later, and at lower CD4+ T-cell counts29. Confirming this assumption, Haas et al. reported switch rates of 3.15/100 (95% CI 2.92—3.40) PY for programs with routine HIV RNA-VL monitoring, 1.21/100 (1.13–1.30) PY for programs with targeted HIV RNA-VL monitoring, and 0.49/100 (0.43–0.56) PY for programs with clinical monitoring. Additionally, some studies have shown that switching following detection of TF can also be delayed due to lack of cART options27. Much of what is detailed in these descriptions applies to treatment programs in Eritrea. Although existing guideline recommend routine VL monitoring, targeted monitoring which depend largely of physician’s discretion and availability of HIV RNA-VL testing reagents is more common. A potential consequence of this approach includes long lag-time between diagnoses of TF and switching31. All in all, improved understanding of the rates of VF, possible lag-time between detection of HIV RNA-VL and switching, and its overall impact on patient’s outcomes are warranted.To support this assertion, they aver that the lack of infrastructure including plasma HIV RNA-VL assays and standard genotypic resistance testing limits the ability to detect TF. Interestingly, a multi-country study involving 300,000 HIV-positive patients noted that rate of switching is largely determined by monitoring strategies—HIV RNA-VL, CD4+ T-cell count, initial functional status, and NRTI or NNRTI used. In the multivariate Cox regression analysis, independent predictors of initial cART modification included residence outside Maekel; late initial WHO clinical stage, certain NRTI and NNRTI. In the adjusted multivariate Cox regression analysis, higher hazards of cART modification were associated with organization, unit reduction in initial weight, residence inside Maekel, initial WHO stage (Stage III), NRTI and NNRTI used. Most of these associations are not unique14. In their report, Njuguna et al. reported the low incidence of drug substitution for TDF-based regimens (2.6 per 100 P/Ys), compared to AZT and d4T based regimens (8.5 per 100 P/Ys vs. 17.9 per 100 P/Ys)32. Separately, substitution rates of 27.0/100 PYs; 1.9/100 PYs; 2.0/100 PYs were recorded for d4T, AZT and TDF-based regimens in a study in Kenya22. Many have suggested that these results highlight the better safety profile of TDF-based backbones22. Similarly, low CD4+ T-cell count has been described as an important predictor of cART modification33. According to some accounts, patients initiating cART at higher CD4+ T-cell count or with WHO Stage I disease are less likely to suffer from AIDS-defining illnesses that require treatment with drugs that may interact adversely with active cART regimens. A similar argument can be applied to the observed link between initial functional status and baseline weight and rate of cART modification. However, the link between address or Organisation and substitution rates remains unclear. This notwithstanding, it is clear that differences in Organization points at quality problems in particular institutions and the need for standardization.In the univariate analysis of factors associated with initial cART modification, substitutions rates differed across a range of factors including hospital of care, baseline weight, address, initial WHO clinical Stage, initial CD48. In practice, however, they noted that the rate of cART modification due to toxicity/intolerance was higher with NVP as compared to EFV8. In this study, Kaplan–Meier analysis of initial regimens demonstrated a higher substitution rate for NVP-based regimes (6.7/1000 (6.3–7.2) PM for EFV-Based regimen vs. 18.6/1000 (17.8–19.4) PM for NVP-Based regimen. However, after adjusting for weight at baseline, NVP-based regimens demonstrated a more favorable retention rate. In the unadjusted Cox regression analysis, NVP-based regimens had a 2.64 (2.45–2.85) hazard of cART modifications, p value < 0.001. In contrast, the adjusted model demonstrated a lower hazard, 0.89(0.81–0.98), p value = 0.018. The likely explanation for this finding is that patients with high baseline weight have better tolerance for NVP-based regimens.Lastly, prior studies have reported conflicting findings on whether NVP-based anchors compared to EFV-based anchors are associated with higher modification hazards. Inzaule et al.reported an incidence rates of 9.80 (95% CI 5.28–18.22) for EFV vs. 7.17 (95% CI 5.58–9.21) for NVPThis study has multiple strengths and limitations. First, the study has a robust data set complete with detailed person-level clinical and demographic data. Secondly, few studies are able to capture long-term treatment modification patterns. In most part, analysis is rarely extended beyond initial regimen and reasons for modifications of partially detailed. In this study, we extended the analysis from the first to the fifth modifications. Consequently, the study provides a more comprehensive picture of cART modification history in four of the largest treatment centers in Eritrea. These strengths notwithstanding, our study has a number of limitations. These include the limitations associated with retrospective studies—missing data problems and non-standardized recordings of key variables.In conclusion, we report the first data describing cART modification and associated factors in Asmara, Eritrea. According to our analysis, most patients have been on NRTI and/or NNRTI. Use of PIs or INSTIs is still limited. The cumulative frequency of all cause first-line cART modification was 3223 (64%). Substitutions were disproportionately higher than switching suggesting possible deficiencies in HIV RNA-VL monitoring. Furthermore, toxicity/intolerance was a leading cause of initial cART modification. However, the impact of toxicity as a driver of cART modification has waned. From the second cART modification onwards, drug shortage was the predominant reason for change. The clinical significance of the high number of shortage-related substitutions at patient level is currently unknown. However, we believe that the outcome points at a significant programmatic gap. In the adjusted multivariate Cox regression analysis, higher hazards of cART modification were associated with treating hospital, unit reduction in initial weight, residence inside Maekel, initial WHO stage, NRTI and NNRTI used.


Adherence barriers to oral regimens are common and span individual and structural factors, including stigma, forgetting, unstable housing, food insecurity, insurance lapses or prohibitive co‐pays, mental illness, substance use, transportation challenges and stock‐outs. Long‐acting (LA) injectable agents administered monthly or less frequently could circumvent some of these barriers.LA agents have been developed for contraception and other disease states like psychiatric illness. For HIV infection, LA treatment is relatively new. The U.S. Food and Drug Administration (FDA) approved a combination of two injectable antiretroviral medications, cabotegravir (CAB) and rilpivirine (RPV), for administration every 4 weeks in both ART‐naïve and ART‐experienced patients in January 2021; a higher dose of each given every 8 weeks was approved in March 2022. The approvals were based on registrational trials of LA CAB/RPV in treatment‐naïve and experienced participants who were first virologically suppressed on oral regimens . In an ucan lead to virologic suppression in unsuppressed patients.All of these studies started LA CAB/RPV in virologically suppressed patients. However, given the difficulties with adherence in multiple contexts worldwide, studies using LA‐ART in adherence‐challenged patients are needed. A demonstration project conducted at the Ward 86 HIV Clinic at San Francisco General Hospital, which serves a publicly insured or uninsured population of people with HIV (PWH) in San Francisco, provides the most extensive experience to date: 133 patients received LA CAB/RPV; 76 (57%) of whom were virologic suppressed on oral ART and 57 (43%) with viremia off of ART due to adherence challenges [Discussions around the use of LA CAB/RPV before and after its approval have centred around the idea of equity and extending the use of this combination treatment to adherence‐challenged and hard‐to‐reach populations, including those with marginal housing, individuals experiencing incarceration and adolescents , 10, 11.The U.S. FDA usually approves medications based on evidence from randomized trials. However, patients with ongoing viremia are often not able to suppress on an oral ART regimen, which can make an RCT difficult to enrol and a likely set‐up for failure in the oral ART arm. A recent modelling study showed that only 22% of patients with long‐standing adherence challenges and viremia are likely to suppress on oral ART, even with wrap‐around services . TherefoIn the case of a “rare condition,” the U.S. FDA allows single‐arm trials to determine the efficacy of a medication in the population . For insAlthough important for market introduction, the drug label is not the only source of evidence for clinical practice and reimbursement. Treatment guidelines from reputable sources can influence which medications are reimbursed. In HIV, the Department of Health and Human Services Panel on Antiretroviral Treatment Guidelines for Adults and Adolescents in the United States  serves tWe thereby recommend the consideration of a well‐conducted, multi‐centre, single‐arm trial examining LA CAB/RPV in viraemic participants. The details of the study design should involve intensive consultation with the community as well as clinicians with expertise in serving these communities to examine safety, efficacy, acceptability and feasibility.Whether or not LA‐ART will change population health outcomes depends on the willingness of the HIV community to test this strategy in viraemic patients in order to generate data for treatment guideline panels and regulatory authorities. To truly reach the U.S. End the HIV Epidemic (EHE) goals, we need a way to harness innovative strategies to help patients with viremia achieve virologic suppression. LA ART can be the “game changer” to help achieve EHE goals, and innovative study designs to rapidly evaluate this important new strategy among viraemic patients are indicated.None of the authors reports any relevant competing interests.MG drafted the original manuscript; JN, VM and TW edited the manuscript, added references and reviewed the final version.