To support optimal third-line antiretroviral therapy (ART) selection in Namibia, we investigated the prevalence of HIV drug resistance (HIVDR) at time of failure of second-line ART. A cross-sectional study was conducted between August 2016 and February 2017. HIV-infected people ≥15 years of age with confirmed virological failure while receiving ritonavir-boosted protease inhibitor (PI/r)-based second-line ART were identified at 15 high-volume ART clinics representing over >70% of the total population receiving second-line ART. HIVDR genotyping of dried blood spots obtained from these individuals was performed using standard population sequencing methods. The Stanford HIVDR algorithm was used to identify sequences with predicted resistance; genotypic susceptibility scores for potential third-line regimens were calculated. Two hundred thirty-eight individuals were enrolled; 57.6% were female. The median age and duration on PI/r-based ART at time of enrolment were 37 years and 3.46 years, respectively. 97.5% received lopinavir/ritonavir-based regimens. The prevalence of nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), and PI/r resistance was 50.6%, 63.1%, and 13.1%, respectively. No significant association was observed between HIVDR prevalence and age or sex. This study demonstrates high levels of NRTI and NNRTI resistance and moderate levels of PI resistance in people receiving PI/r-based second-line ART in Namibia. Findings underscore the need for objective and inexpensive measures of adherence to identify those in need of intensive adherence counselling, routine viral load monitoring to promptly detect virological failure, and HIVDR genotyping to optimize selection of third-line drugs in Namibia. Thus, comparably less information is available to guide selection of optimal regimens in people with virological failure while taking second-line ART.Global HIV drug resistance (HIVDR) surveillance efforts and the majority of studies performed in low- and middle-income countries have focused on estimating the prevalence of HIVDR prior to treatment initiation or after failure of first-line NNRTI-based therapies.ART in Namibia is delivered following a public health approach, which involves use of standardized first- and second-line regimens and simplified laboratory monitoring, including at least one viral load test per year. At the time of study enrolment, Namibia's national ART guidelines recommended first-line regimens consisting of two nucleoside reverse transcriptase inhibitors (NRTI), tenofovir (TDF)/emtricitibine (FTC) or lamividuine (3TC) administered with the NNRTI efavirenz (EFV). Recommended second-line regimens were three NRTI , or abacavir (ABC)) administered with a ritonavir-boosted protease inhibitor, either lopinavir/ritonavir (LPV/r) or atazanavir/ritonavir (ATV/r).At time of study initiation (2016), 140,241 adults out of an estimated 204,147 adults living with HIV were receiving ART in Namibia of which 3884 were taking PI/r-based second-line regimens . Globally and in Namibia, unanswered questions remain about the contribution of protease inhibitor (PI) drug resistance to second-line ART failure. In this study, we report the prevalence and patterns of HIVDR in people failing second-line ART in Namibia's public health ART program.22.1The 15 ART clinics with the largest number of people on PI/r-based ART in the country were selected. These 15 clinics captured 70% (2746 of 3884) of all people receiving PI/r-based ART; clinics were located in nine different geographical regions: Khomas, Ohangwena, Zambezi, Oshikoto, Oshana, Kavango East, Erongo, Omusati, and Otjozondjupa.Between August 2016 and February 2017, all HIV-infected people ≥15 years of age receiving second-line PI-based ART for at least six months and who had confirmed virological failure per Namibia national ART guidelines (two consecutive HIV RNA tests ≥1000 copies/mL separated by at least three months) were identified and asked to participate in the study during routine clinic visits. Written informed consent was obtained from all participants. Individuals with self-reported treatment interruption of 30 days or more at the time of enrolment were ineligible; no additional information was collected about this group.2.2 and were frozen at –80oC for storage. DBS from participants who had a third consecutive VL test ≥1000 copies/mL were brought to ambient temperature, re-packed into new gas impermeable bags with desiccant and humidity indicator cards and shipped to South Africa for HIVDR testing.At study sites, nurses drew 5 mL of whole blood via venepuncture for viral load (VL) testing (a third consecutive VL test). Whole blood specimens were transported in a cold box on the day of collection to the Namibian Institute of Pathology (NIP) for VL testing. On arrival at NIP, dried blood spot (DBS) specimens were prepared by pipetting 75 μL aliquots of whole blood on each of 5 circles of a Whatman 903 filter paper card . DBS were dried at room temperature for 24 hours and subsequently packed in individual gas impermeable bags per World Health Organization (WHO) guidanceVL testing was performed on plasma within 48 hours of specimen receipt using COBAS AmpliPrep/COBAS TaqMan HIV-1 Test . HIVDR testing was performed at the National Health Services Laboratory, University of Witwatersrand, Johannesburg, South Africa, a WHO-designated HIVDR genotyping laboratory. RECall was used to generate consensus sequences; quality assurance was performed following WHO recommendations. The Stanford HIV drug resistance algorithm (HIVdb), version 8.4, was used to identify HIVDR resistance profiles, mutations, and HIV subtypes; sequences classified as having low-, intermediate-, or high-level HIVDR were considered as resistant. The HIV-1 integrase region was not sequenced because at the time of this study integrase strand transfer inhibitors (InSTI) were not prescribed for treatment of HIV in Namibia's public health sector.HIV-1 protease and reverse transcriptase (RT) sequences were obtained using standard population sequencing adapted from previously described methods.,10 The algorithm was used becasue Namibia does not have an established algorithm for selection of third-line ART based on HIVDR genotype test results. Genotypic susceptibility scores (GSS) for individuals who would have been eligible for third-line ART  were calculated based on HIVdb penalty scores for each drug in the regimen that would have been prescribed following the algorithm. The ARV drugs used in the algorithm include darunavir/ritonavir (DRV/r), 3TC/FTC, ZDV or TDF , and an InSTI if the TDF or ZDV score is 30 or above or the DRV/r score is 15 or above. In addition, etravirine (ETR) is added if the TDF or ZDV score is 30 or above and the DRV/r score is 15 or above and the ETR score is below 30. One GSS point was given for each drug with a Stanford HIVdb penalty score <15; it was assumed that virus was fully susceptible to all approved InSTI.Potential third-line treatment options were considered based on an algorithm developed by South Africa's national third-line ART committee.2.3 Demographic and laboratory characteristics are summarized as prevalence values or median values and interquartile ranges (IQR). HIVDR prevalence is reported with 95% Wilson confidence intervals adjusting for site-level clustering using a robust standard error. Fisher's exact tests and Wilcoxon rank sum tests were used to conduct two-group comparisons with a significance level of 0.05.Analyses were performed using Stata .2.4The study received institutional review board (IRB) approval from the Ministry of Health and Social Services in Namibia and expedited review from the IRBs of Tufts University School of Medicine, the University of Witwatersrand, Johannesburg, South Africa. The study was reviewed in accordance with the Associate Director for Science at the Center for Global Health, United States Centers for Disease Control and Prevention CDC human research protection procedures and was determined to be research. CDC investigators did not interact with human subjects or have access to identifiable data or specimens for research purposes.3All 238 eligible individuals identified during the study period were enrolled; 96.6% (230/238) were receiving a regimen consisting of three NRTIs in combination with either ATV/r or LPV/r. Table HIVDR genotyping was successful for 160/238 (67.2%) participants. Overall 70.0% (112/160) of sequences had mutations conferring drug resistance to any drug; 50.6%, 63.1%, 13.1%, and 6.9% had any NRTI, any NNRTI, LPV/r or ATV/r, DRV/r resistance, respectively  had three or more major PI mutations Table . In addi,11 are presented in Table GSS scores for potential third-line regimens following guidance developed by South Africa's national third-line ART committee4This study of HIVDR in people failing PI/r-based second-line ART in Namibia has important findings. First, 30% of people failing PI/r-based ART had no predicted HIVDR and only 13% had PI resistance, suggesting that most virological failures were not due to drug resistant virus. This observation suggests that greatly intensified adherence support may be an optimal initial approach to patient management of viral non-suppression in people receiving PI/r ART in Namibia. Secondly, our findings support the importance of routine VL monitoring for early detection of suboptimal adherence, followed by HIVDR testing in individuals identified as failing therapy, an approach which is likely to minimize premature switch to more complex and costly third-line regimens.–14In this study, overall NRTI resistance levels were high and could lead to reduced efficacy of the NRTI backbone for half of the people evaluated for third-line PI/r-based regimens. However, the comparably low prevalence of PI resistance observed in this study is reassuring given that genotypic testing may not accurately predict regimen activity when providing PI/r-based ART after second-line PI failure: PI/r-based regimens have demonstrated efficacy in people with NRTI resistance, even when predicted NRTI resistance is complete, suggesting that NRTIs retain partial antiretroviral activity or that NRTI-resistant viruses have reduced fitness.Thirteen per cent of study participants failed with PI-resistant virus, emphasizing the need for HIVDR testing in people receiving PI/r-based second-line treatment to ensure the selection of highly potent third-line regimens  and to identify individuals with virological failure due to suboptimal adherence  who are likely to benefit from enhanced adherence counselling. Although 87% of patients in this cohort have no PI resistance, informal patient surveys in Namibia's national ART programme suggest that the majority of people failing PI/r-based ART report inconsistent adherence to the PI component of the regimen due to PI-specific toxicity or side effects; thus resulting in dual therapy . Because of expense and logistics, HIVDR genotyping remains a costly adherence test, presenting an additional challenge: the lack of robust and inexpensive objective measures adherence to ART and specifically the PI/r component, which may lead to delayed switch in the face of true HIVDR or premature switch in people with no HIVDR.–17 and in a recent retrospective non-representative convenience sample of 366 HIVDR genotypes obtained by Namibia's ART program during the period 2010 to 2015.The prevalence estimates of overall, drug class and drug-specific resistance observed in this study are broadly comparable to those observed in other countries In addition, in a recent meta-analysis, DRV/r monotherapy in ART-experienced people was shown to be effective. However, one-third (7 of 21) of people in this Namibian cohort would have been treated with regimens having suboptimal predicted efficacy because their viruses were fully susceptible to the InSTI only. Despite its low propensity to select for drug resistance mutations, there are an increasing number of reports of virological failure associated with InSTI resistance in ARV drug-experienced InSTI-naïve individuals receiving a dolutegravir-containing regimen,,21 underscoring the need for caution and close virological monitoring of this sub-population who may be at risk of virological non-suppression.The predicted activity of potential third-line regimens according to South Africa's national algorithm is somewhat encouraging, since most  patients were either susceptible to DRV/r or had at least two active drugs available. A recent publication assessing the efficacy of South Africa's third-line algorithm showed a high proportion of individuals on third-line had achieved HIV viral suppression  after a minimum of six months; therefore these results may not be generalizable to populations failing WHO-recommended second-line ART regimens consisting of only two NRTIs and PI/r. Nonetheless, our findings reflect the majority of those adults failing second-line ART in Namibia and provide important information that can be used programmatically for the management of people experiencing virological failure of PI/r-containing regimens in the country.Medication non-adherence appears to be an important cause of second-line ART failure in one third of people in this study. A limitation of this study is that pharmacy refill data, pill count, or plasma, hair, or urine drug levels were unavailable; therefore, objective measures of adherence could not be assessed. This study has additional limitations. Although study sites comprised over 70% of all people receiving PI/r in Namibia at the time of the study, sites may have differed from other regions in the country with respect to patient demographics or ARV drug treatment histories by virtue of their larger patient populations. Although amplification success rates were similar across all clinics, there is a possibility that specimens that failed to amplify may have more or less drug resistance than those that amplified; however, as described, there was no differences between people with and without genotypes available for analysis. In addition, nearly all study participants were receiving three NRTIs in addition to PI/r (as per Namibia guidelines5This study demonstrates high levels of NRTI resistance and moderate levels of PI resistance in people receiving PI/r-based second-line ART, supporting the need to optimizing treatment adherence to all three second-line ARV drugs, use of routine VL monitoring to promptly detect virological failure, and HIVDR genotyping to optimize selection of third-line drugs. In addition, programmatic concerns about selective non-adherence to PI/r argue for more tolerable highly potent and fixed-dose combination regimens.MRJ and SYH conceived the study. MRJ wrote the protocol, designed the study and wrote the first draft of the manuscript. NH, LB, SS, SA, ANS, TN, CLF-W, EGR, NM, CJH, and AMT supported implementation of the study and interpretation of data in light of Namibia's public health ART Programme. ND performed statistical analysis. KS, VK, SC perofmred HIVDR genotyping and quality assurance of sequences. MRJ and NP performed additional sequence quality assurance and performed the HIVDR interpretation and genotype susceptibility scoring. All authors contributed to the final draft of the manuscript and had access to the data.


Expanded access to combination antiretroviral therapy (cART) throughout sub-Saharan Africa over the last decade has remarkably improved the prognosis of persons living with HIV (PLWH). However, some PLWH experience virologic rebound after a period of viral suppression, usually followed by selection of drug resistant virus. Determining factors associated with drug resistance can inform patient management and healthcare policies, particularly in resource-limited settings where drug resistance testing is not routine.A case–control study was conducted using data captured from an electronic medical record in a large treatment program in Nigeria. Cases PLWH receiving cART who developed acquired drug resistance (ADR) and controls were those without ADR between 2004 and 2011. Each case was matched to up to 2 controls by sex, age, and education. Logistic regression was used estimate odds ratios (ORs) and 95% confidence intervals (CIs) for factors associated with ADR.We evaluated 159 cases with ADR and 299 controls without ADR. In a multivariate model, factors associated with ADR included older age , higher education level (secondary OR 2.14 [95% CI 1.1.11–4.13]), compared to primary and tertiary), non-adherence to care (OR = 2.48 [95% CI 1.50–4.00]), longer treatment duration (OR = 1.80 [95% CI 1.37–2.35]), lower CD4 count((OR = 0.95 [95% CI 0.95–0.97]) and higher viral load (OR = 1.97 [95% CI 1.44–2.54]).Understanding these predictors may guide programs in developing interventions to identify patients at risk of developing ADR and implementing prevention strategies. Acquired and transmitted antiretroviral drug resistance mutations among persons living with HIV (PLWH) are a major public health concern, as they can limit the efficacy of available drugs for the management of HIV . Resista3) ), higher education level , longer treatment duration (OR = 1.80 [95% CI 1.37–2.35]), and higher viral load (OR = 1.97 [95% CI 1.44–2.54]) remained significantly associated with ADR  ADR. These findings somewhat confirm, and at times diverge, from what has been described previously as factors associated with ADR. Although Khienprasit et al.  reportedMarried people usually have more family support so adherence to ART can be better handled or prevented through being reminded by the spouse or other family members. Marital status has been found to influence health and mortality, and give a lot of stability. Kiecolt-Glaser and Wilson  in theirAnother major predictor of ADR was education. However, when adjusted for confounders, only secondary education remained statistically significant. Two different studies, one in Southeast Nigeria, and another in Upper West Region of Ghana , 39 repoThe mean treatment duration was significantly different between those that developed ADR and those that did not. This result is not surprising. In the early part of the ART program in Nigeria there were months of drug stock-outs, lack of adequate supply management of medical commodities, weak laboratory infrastructure, and conditions that may favor the occurrence of high levels of ADR. Monitoring of treatment was only done by CD4 cell count and hardly viral load and genotype testing. Patients would therefore have been maintained on virologically failing regimens while multidrug resistant viruses accumulated and thereby made available drugs ineffective over time. In a comparable study in Tanzania by Asgeir et al.  the emerIncidentally, baseline CD4 cell count prior to ART initiation regarded as the most significant predictor of survival after initiation of first ART, was not strongly linked to the development of drug resistance. However, the CD4 count at development of ADR was very significant. The association between CD4 cell count and drug resistance has a biological reason. In this study, however, the analyses may have been partly confounded by the fact that most of the patients were enrolled on treatment with very low CD4 cell counts. Uy et al.  and JoseThe main limitation in this study was that data extracted from routine medical records may have been incomplete, inconclusive, or inaccurate. Additionally, it was difficult to know which factor acted first in the development of ADR in a patient who had several identified predictors. For the purposes of this study an association design was appropriate despite its limitations because the intention of this study was to determine if a relationship existed between the predictors and ADR.The predictors outlined in this study should be recognised among vulnerable populations by health care providers especially in the resource-limited settings. It becomes critical to educate patients of possible onset of ADR especially those who are vulnerable based on these identified predictors. Policy makers, social advocacy groups and Health Ministries would use the information and be more focused in treatment and deploy resources to managing many more patients on first-line drugs instead of few on the much more expensive, scarce, second and third-line drugs due to the development of ADR.


The aim of this study was to assess trends in drug resistance and associated clinical and programmatic factors at a national level during the rapid scale up of ART.Logistic regression was used to identify the factors associated with HIVDR. Variables associated with drug resistance in multivariable logistic regression were included in the Cochran–Armitage test for trend.P < 0.01). With respect to the class of antiretroviral, there were substantial increases in resistance to both non-nucleoside reverse transcriptase inhibitors (NNRTIs) and nucleoside reverse transcriptase inhibitors (NRTIs) . The prevalence of DR to protease inhibitors (PIs) was low, which supported their continued use as second-line therapy in China.A total of 11,976 patients were enrolled in the study. The prevalence of HIVDR among patients who received ART for 9–24 months during 2003–2008, 2009–2012, and 2013–2015 significantly decreased (15.5%, 6.3%, and 2.3%, respectively, Our results provide evidence for the effectiveness of China’s “Treat All” approach to guide policy makers to improve training for healthcare providers and education on ART adherence among patients. The global expansion in the use of antiretroviral therapy (ART) has markedly reduced HIV-related morbidities, mortalities, disease transmission, and adult HIV incidence ,2. HowevWith the continued roll-out of ART in LMICs, evidence is emerging of increasing levels of drug resistance prevalence , 6. To eThree phases of ART were established according to development stages of NFATP in China: initial and scale-up ART, 2003–2008; standardization and scale-up, 2009–2012; and rapid scale-up, 2013–2015 [Data collected through the Chinese National HIVDR Surveillance and Monitoring Network since 2003 were analyzed using a retrospective method to assess HIVDR and associated program factors in populations of patients on ART. The database included annual cross-sectional surveys of HIV drug resistance from 2003 to 2015 in China which were described in the previous studies , 16–18. http://hivdb.stanford.edu/, accessed 2 November 2018). All HIVDR mutations that conferred low , intermediate  or high-level  resistance were included [Blood samples of the participants were collected by the local CDC at enrolment and every follow-up visit. Within 8 h, blood samples were sent at ambient temperature to the laboratory of NCAIDS in Beijing, where a team was on-call 24 h for 7 days a week for receipt. Within 2 h after arriving at the laboratory, CD4 cell count was conducted using flow cytometry . Plasma was isolated and frozen at − 80 °C until testing for viral load and HIVDR. According to the manufacturers’ recommendations, plasma HIV RNA in this study was quantified with real-time NASBA  or with Amplicor HIV-1 monitor test . A VL ≥ 1000 was classified as virologic failure based on WHO HIVDR surveillance guideline. In samples with virologic failure on ART, HIVDR genotyping was performed at the WHO Accreditation Laboratory of NCAIDS, China CDC by using an in-house method as previously described , 20, 21.included . The freP value < 0.05. All the statistical analyses were performed with SAS 9.4 software .Logistic regression was used to identify the factors associated with HIVDR. The factors associated with HIVDR that were significant in the univariate logistic regression analysis were included in the multivariate logistic regression. Variables associated with resistance in the multivariable logistic regression model and those clinically meaningful were included in the Cochran–Armitage test for trend. Somers’ D (R|C) was used to measure the association between the variables (considered as the response) and the time of initial ART . Statistical significance was defined as a P < 0.01). The factors significantly associated with HIVDR in the multivariate logistic regression model were education, occupation, route of HIV infection, WHO clinical stage III/IV, CD4 cell count before ART, and time of beginning ART. Compared with patients with post-secondary school or higher education, those with secondary school or lower education were more likely to show DR . Compared with people from other occupations, farmers were more likely to show DR . With regard to route of HIV infection, compared with heterosexual intercourse, homosexual intercourse was likely to show less DR ; blood or plasma transfusion was more likely to show DR ; intravenous drug use was more likely to show DR ; and other or unknown routes were more likely to show DR . Furthermore, compared with patients with other WHO clinical stages, those with WHO clinical stage III/IV were more likely to show DR . Patients with CD4 count ≥ 349 cells/mm3 showed less DR than patients with CD4 count < 349 cells/mm3 , and patients who began ART from or after 2009 showed less DR than patients who began ART before 2009 . With regard to the location of ART drug distribution, county hospital or CDC showed lower DR than township hospital or village clinic . Patients who missed any dose in the past month were more likely to show DR than those who did not miss any dose . Compared with lamivudine-based regimens, other regimens were more likely to show DR   patients had HIVDR. The prevalence of HIVDR during 2003–2008 n = 1882), 2009–2012 (n = 1163), and 2013–2015 (n = 1634) significantly decreased . The most-common NNRTI mutations occurred at positions 101 (9.5%), 103 (49.5%), 181 (12.9%), and 190 (9.5%) in the RT region; the most-common NRTI mutations occurred at codons 41 (10.2%), 67 (13.4%), and 184 (32.3%) in the RT region; and protease inhibitors (PI) mutations were most frequently seen at codons 32 (0.7%), 46(1.5%) and 54 (0.7%) in the protease (PR) region . The recent  prevalence of HIVDR among patients who received first-line ART for 9–24 months was found to be 6.3 and 2.3%, respectively. The prevalence of HIVDR found in our study was significantly lower than that in some developed countries or countries where free ART was available nationwide . The dru3; this threshold then increased to 350 cells/mm3 in 2012 and further increased to 500 cells/mm3 in 2014. A further important policy change in NFATP included the location of ART drug distribution; increased rates of distribution at county hospitals or CDC have occurred over time, which has improved the quality of care and treatment. The stratified analysis showed that patients receiving ART at county hospitals had lower HIVDR rates than those treated at village- or town-level clinics.The overall low prevalence of DR may reflect the relatively low coverage and short duration of free ART or, equally, may reflect the success of first-line ART regimens and HIV prevention programs. Our study assessed the clinical and programmatic factors associated with HIVDR in patients who received first-line ART during 2003–2015. Virologic failure among Chinese patients who received ART for 9–24 months during 2003–2008, 2009–2012, and 2013–2015 significantly improved .The significant improvements in virologic outcome in Chinese patients receiving ART treatment over the past 10 years may be attributable to changes in a number of clinical and programmatic factors. Firstly, the Chinese NFATP policy has changed since 2003, due to the availability of an expanded range of antiviral drug resources. For instance, AZT, d4T, ddI, and NVP were generically produced in China and used as standard prescriptions from the beginning of the NFATP, whereas 3TC and EFV were branded drugs that became available in 2005 and were gradually more commonly prescribed. First-line ART regimens were revised to consist of tenofovir (TDF)/AZT + EFV/NVP in 2008 [Changes in social and demographic factors among patients since the initiation of NFATP in 2003 had also been associated with treatment outcome over time. The proportion of HIV patients with a higher education level (high school and above) increased from 14.5% in 2003–2008 to 43.7% in 2013–2015. In addition, more HIV patients were from non-agricultural occupations (increased from 30.1 to 53.8%) and the number that had contracted HIV due to heterosexual or homosexual intercourse, compared to other routes such as drug use, increased from 31.6 to 53.0%  in China. Among the 402 treatment-experienced patients with DR mutations, 215 (53.5%) harbored dual-class resistance. With respect to the class of ART drugs, there were substantial increases in resistance to both NNRT and NRTIs over time (from 49.7 to 73.0%). In particular, expanded ART without improved adherence is likely to result in dual-class resistance, which may lead to poor treatment outcomes and HIVDR. The most common NNRTI mutations were K103 and Y181, and the most common NRTI mutations were M184, D67 and M41. In addition, the prevalence of DR to PIs was low, which supported their continued use as second-line therapy in China.Apart from important findings, our study has several limitations. First, unlike a cohort study, in which a group of patients is assessed continuously over time, a cross-sectional survey, by its very nature, excludes patients who are no longer receiving ART and therefore cannot be observed because they have died, been lost to follow-up, or have stopped treatment. This survivor bias can significantly impact the interpretation of outcomes. In order to adjust for such a possible bias, we collected nationally representative data on retention at 12 months. By the end of 2015, 10.2% of the patients had died, whilst 8.7% were found to have terminated treatment among those treated by the NFATP in China. Accordingly, the adjusted prevalence of virologic failure and DR among patients who received first-line ART for 9–24 months during 2013–2015 was 7.0–8.3 and 2.3–2.7%, respectively. Second, a large proportion of patients began initial ddI treatment during the earlier years of the NFATP, when the baseline CD4 cell count was not required to be of a certain threshold and the standards of care and follow-up were low, which might have contributed to the poor treatment outcomes in the first few years of the NFATP.However, these limitations do not have a considerable bearing on our conclusion on the changes in HIVDR from 2003 to 2015 in China. To our knowledge, this is the first large observational study worldwide to assess the outcomes of ART over the past 10 years. The results provide evidence for the effectiveness of China’s HIV control and prevention efforts such as the “Treat All” approach. Furthermore, the findings will help guide policy makers to improve training for healthcare providers and enhance patient education on ART adherence. Further monitoring of virologic outcomes is needed to elucidate the determinants of long-term programmatic success. Government advocacy, specialist physician management, laboratory monitoring and constantly raising the level of education, all played a role in the success of NFATP. This study had the advantage of having thoroughly examined these factors and their relationship in one analysis, and the results were particularly important for countries with limited resources.


There are few data on the prevalence of acquired drug resistance mutations (ADRs) in Hunan Province, China, that could affect the effectiveness of antiretroviral therapy (ART).The main objectives of this study were to determine the prevalence of acquired drug resistance (ADR) the epidemic characteristics of HIV-1-resistant strains among ART-failed HIV patients in Hunan Province, China.HIVdb.stanford.edu algorithm to infer drug susceptibility.ART-experienced and virus suppression failure subjects in Hunan between 2012 and 2017 were evaluated by genotyping analysis and mutations were scored using the The prevalence of HIV-1 ADR were 2.76, 2.30, 2.98, 2.62, 2.23and 2.17%, respectively, from 2012 to 2017. Overall 2295 sequences were completed from 2932 ART-failure patients, and 914 of these sequences were found to have drug resistance mutation. The most common subtype was AE (64.14%), followed by BC (17.91%) and B (11.50%). Among those 914 patients with drug resistance mutations,93.11% had NNRTI-associated drug resistance mutations, 74.40% had NRTI drug resistance mutations (DRMs) and 6.89% had PI DRMs. Dual-class mutations were observed in 591 (64.66%) cases, and triple-class mutations were observed in 43 (4.70%) cases. M184V (62.04%), K103N (41.90%) and I54L (3.83%) were the most common observed mutations, respectively, in NRTI-, NNRTI- and PI-associated drug resistance. 93.76% subjects who had DRMs received the ART first-line regimens. CD4 count, symptoms in the past 3 months, and ART adherence were found to be associated with HIV-1 DR.This study showed that although the prevalence of HIV-acquired resistance in Hunan Province is at a low-level, the long-term and continuous surveillance of HIV ADR in antiretroviral drugs (ARVs) patients is necessary. HIV has been a leading cause of infectious diseases and death globally over the last three decades. Since highly active antiretroviral therapy (HAART) became available, the HIV-infected patients’ life expectancy and health outcomes have been drastic improved. However, the widespread use of HARRT has led to drug resistance (DR) to ARVs, which undermines their effectiveness , 2. DrugSince the first case of HIV infection was found in 1992, there were 29,002 people living with HIV (PLWH) to the end of 2017 in Hunan province, which is in south central China, and has a population of 68 million. More than 25,530 HIV/AIDS patients had received free highly active antiretroviral therapy (HAART) and were still in treatment which is supported by the government by the end of 2017. Although stavudine (D4T) and zidovudine (AZT) are not recommended as first-line therapy in the US and Europe, in much of Asia and other resource-limited settings, the most common first-line regimens for HIV-infected patients still contain either D4T or AZT, which are less costly than tenofovir (TDF) –12.The emerging ADR and subsequent treatment failure pose a major concern for HIV ART in resource-limited settings where treatment options are limited. The objective of this study is to determine the prevalence and DRMs of HIV-1 virologic failure and acquired drug resistance among ART-experienced adults in Hunan Province, China.The research protocol, approved by the relevant institutional review boards or independent ethics committees, was conducted in accordance with standards for the protection of patient safety and welfare and in compliance with Good Clinical Practices and the principles of the Declaration of Helsinki and its amendments. According to “National Free AIDS ARV guidelines” (China), all participants who receive treatment from AIDS ARV supported by the Chinese government must have viral load testing and genotypic testing, which will be used for this study. Verbal informed consent is given to the doctors at the clinical sites who are responsible for the AIDS patients, and the patients’ names were kept on a list with the doctors’ signature. The study and the verbal consent procedure were approved by the Ethical Committee of Hunan Provincial Center for Disease Control and Prevention .The Chinese national free ARV treatment policy guidelines recommend that patients on ART should have an HIV viral load test at least once a year –12. If t+ T cell abs counts were determined with a FACS Calibur  in freshly collected whole blood (within 24 h). Plasma HIV-1-RNA viral loads were determined using the COBAS TaqMan HIV-1 test, version 2.0 (CAP/CTM v2.0) with a lower limit of detection of 40 RNA copies/ml and Abbott m2000 Diagnostics Systems with a lower of detection of 150 RNA copies/ml.CD4st round was implemented using Taq PCR Mastermix  with primers: PRT-F2(5′- CTTTARCTTCCCTCARATCACTCT − 3′) and RT-R2(5′-CTTCTGTATGTCATTGAC AGTCC − 3′) and the condition were: 94 °C for 4 min, then 30 cycles of 94 °C for 15 s,55 °C for 20 s, and 72 °C for 2 min and then 72 °C for 10 min. The PCR products were purified and sequenced by the North Genomics Research Center .HIV RNA was then extracted from 140 ul of plasma using the QIAmp Viral RNA mini kit  according to the manufacturer’s protocol. Approximately 1247 bp pol fragments (HXB2 positions 2057–3304) were amplified by nested reverse transcriptase polymerase chain reaction (nested RT-PCR). The 1st round PCR using one-step RT PCR kits  with primers: F1a (5′- TGAARGAITGYACTGARAGRCAGGCTAAT − 3′), F1b(5′- ACTGARAGRCAGGCTAATTTTTTAG − 3′) and RT-R1(5′-ATCCCTGCATAAATCTGACTTGC − 3′) and the cycling conditions were as follows: incubation at 50 °C for 45 min, 94 °C for 2 min, then 35 cycles of 94 °C for 15 s, 50 °C for 20 s, and 72 °C for 2 min 30 s, the 72 °C for 10 min. The 2 http://hivdb.stanford.edu), with a resistance score of ≥15 for any drug [P < 0.05 were used to determine statistical significance.The results from sequencing were aligned and assembled manually by Bio Eidt (7.0.2) and ContigExpress Project, a component of the Vector NTI Suite 6 software, Major drug-resistance mutations were defined according to the IAS-USA 2015 list . The subany drug , 15. StaFor the years 2012 to 2017, there were 1520, 3650, 6749, 8215 and 9380 patients respectively who received ART more than 6 months and who had done the VL testing. The proportion of patients with ART failure (VL >1000copies/ml) was 8.55% (130/1520), 8.30% (303/3650), 8.67% (585/6749), 8.23% (675/8215), 7.49% (703/9380) and 7.12% (536/7524).Most of the subjects were male . The median age was 44. (IQR: 33–52), and the age distribution was mainly in adults aged 30–44 years old, but the percentage of the older population (> 60) has increased year by year (from 12.31 to 25.19%). Half of the study population were married or cohabiting . The most common route of infection was heterosexual , the proportion of PWID (persons who inject drugs) showed a declining pattern each year from 2012 to 2017.The median virus load of participants was 18,882 copies/ml , and the median CD4 was 189 cells/ml (IQR:81–277). (Table pol area complete sequences (1200 bp) were obtained by Sanger sequencing. The primary subtype was AE , followed by BC  and B  . Y181C also emerged frequently in subtype CRF01AE sequences .Nine hundred and fourteen of 229539.83%) had at least one primary drug resistance mutation (DRM) according to the Stanford HIVdb algorithm SDRM list of mutations . The ann9.83% hadEight hundred and fifty-seven of 914 subjects (93.76%) who had DRMs received the ART first-line regimens that include two NRTIs and one NNRTIs. All the regimens contain 3TC, 468 patients took AZT + NVP/EFV, 102 took D4T + NVP/EFV and 287 took TDF + NVP/EFV. 6.13% of patients (56/914) received the second-line regimens which were made with LPV/r and two NRTIs /NNRTIs. Only 1 patient took the Raltegravir (RAL) + 3TC+ Abacavir (ABC). Among the 1381 no DRMs subjects, 882 took the first-line regimens: 424 took AZT + NVP/EFV, 62 took D4T + NVP/EFV and 396 took TDF + NVP/EFV; 499 received the second-line regimens (LPV/r or RAL or ABC + 3TC+ NVP).p < 0.05). The odds of having a drug resistance mutation were 3.15 times higher for a patient who had low-level CD4 count(≤200 cells/mm3) than for a patient who had a high-level CD4 count(> 200 cells/mm3)(95% CI:2.54–3.89). The patients who had symptoms in the last 3 months had a higher risk for developing drug resistance mutation compared with those with no symptoms in the last 3 month . There were 59.63% (545/914) DRMs detected subjects who reported missing a dose in the last 7 days, and compared to 37.29% (515/1381) no mutation detected subjects who missed a dose in the last 7 days . The patients taking regimens containing TDF or LPV/r had fewer resistance mutations compared with the patients on regimens containing AZT (P < 0.05). The age, gender, subtype, WHO clinical stage and routes of infection did not show significantly differences between DRMs identified and no DRM identified group (P > 0.05).Nine potential risk factors were considered in the multivariate logistic regression analysis Table . Of thesOur study aimed to elucidate the prevalence of HIV ADRs in ART VF subjects, since ADR is one major problem with the efficacy of ART as it affects the clinical outcomes of treatment. In this study, the annual ADR rate was lower than the threshold low-incidence rate defined by WHO of 5%  . This may indicate a more complicated and diverse trend of HIV-1 epidemiology emerging in Hunan province.The most frequently identified ADR present in NRTI was M184V/I (61.27%), followed by K65R (19.96%). The elevated presence of M184V/I is expected and arises as a consequence of the use of 3TC as part of all the first-line regimens in China . AlthougNNRTIs are notorious for rapidly triggering the emergence of drug-resistant HIV-1 variants. One NNRTI drug mutation can lead to multiple and high-level NNRTI-associated drug resistance . The preThe overall rate of resistance to PIs was low (7.44%) in our study. This might be explained by the low PI coverage in China as a second-line regimen; and since PIs have a much higher barrier to resistance, patients receiving PI-based regimens are less likely to develop resistance. Our study results showed increasing trends in PIs mutations year by year from 2012 to 2017(from 0 to 8.31%), associated with the increasing use of second-line regimens in recent years. Therefore, it is advisable to include surveillance of PIs mutation in the coming years.3), clinical symptoms and bad medication compliance (missing drugs) could more easily lead to HIV-1 drug- resistance mutation among participants experiencing treatment failure. These phenomena are a reminder that monitoring resistant strains using CD4 counts and clinical symptoms in the course of ART as well as evaluating patients’ therapy compliance are very important. TDF has been commonly used as the first-line regimen since 2014, and LPV/r has always been used as the second-line regimen. The regimens containing TDF or LPV/r resulted in less drug resistance compared with AZT-contained ARVs because they were not used as much as AZT in Hunan Province. But this study result showed that the PI related mutation had an tendency to increase year by year from 2012 to 2017. This means that in the future we should pay more attention to second-line regimens drug resistance surveillance.Our study results revealed that the patients’ low-level CD4 count  was relatively low (free VL testing once a year) due to local government policy, all of which may have reduced our power to detect associations between ADR and virologic outcome.In contrast with our previous research, we focused on HIV ADR in Hunan Province during the last 6 years of this study. Although free antiviral therapy has been widely practiced in Hunan Province for more than 15 years, the prevalence of HIV-acquired resistance in Hunan Province is at a low-level. However, the resistant strains are common among those with viral suppression failure. Therefore, long-term and continuous surveillance of HIV ADR in ARV patients is necessary.


Pneumocystis pneumonia (PCP) is a common acquired immune deficiency syndrome (AIDS)-related opportunistic infection. Recent reports estimate that more than 400,000 patients with human immunodeficiency virus (HIV) develop PCP each year globally. However, the timing of antiretroviral therapy (ART) initiation for HIV-infected patients with PCP is still controversial, and the benefits and risks of early initiation of ART are not completely clear. We thus designed this study in order to determine the optimal timing for ART initiation for HIV-positive patients with moderate to severe PCP.This study will be an open-label, multi-centre, prospective randomised controlled trial. A total of 200 subjects will be randomly assigned to an early ART initiation group (≤14 days after PCP diagnosis) and a deferred ART initiation group (>14 days after PCP diagnosis) at a 1:1 ratio. All subjects will be followed up for 48 weeks after starting ART. The primary endpoint is incidence of disease progression (including new or relapsing opportunistic infections and death) at week 48. The secondary endpoints are the changes in CD4 counts from baseline at weeks 12, 24 and 48; the degree of virological suppression (HIV RNA < 50 copies/mL) at weeks 24 and 48; the rate of development of PCP-associated immune reconstitution inflammatory syndrome; and adverse events over 48 weeks.We hope that the results of this study will reveal the optimal timing for initiation of ART in HIV-infected patients with moderate to severe PCP.chictr.org.cn on February 1, 2019. The registration number of the general project is ChiCTR1900021195.This trial was registered as one of the 12 trials under the name of a general project at  Pneumocystis pneumonia (PCP), one of the most common acquired immune deficiency syndrome (AIDS)-defining diseases, is caused by the fungal opportunistic organism Pneumocystis jirovecii and has been effectively controlled in patients with AIDS by the widespread use of modern antiretroviral therapy (ART). However, the incidence of PCP among patients with undiagnosed human immunodeficiency virus (HIV) increased from 48% in 2000 to 67% in 2013 [Optimal timing for ART initiation in opportunistically infected patients with HIV infection is controversial. One multi-centre randomised clinical trial showed that early initiation of ART had a lower rate of AIDS progression and deaths than deferred ART, and adverse events (AEs) did not increase and viral suppression was optimal . In the This study aims to investigate the optimal timing for ART initiation in HIV-infected patients with moderate to severe PCP. Our primary goal is to compare the progression of disease between an early ART initiation group (initiation within 2 weeks of PCP diagnosis) and a deferred ART initiation group (initiation after 2 weeks of PCP diagnosis) at week 48. Our secondary goal is to compare the safety of the timing of ART initiation between the early ART initiation group and the deferred ART initiation group during the 48-week period of this study. Our tertiary goal is to determine whether there are differences in the long-term effects of early ART initiation as compared with deferred ART initiation with regard to CD4 cell counts and HIV RNA loads in HIV-infected patients with moderate to severe PCP.This study will be conducted as an open-label, multi-centre, prospective randomised controlled trial. We will recruit 200 subjects from the following 17 hospitals: Chongqing Public Health Medical Center, Beijing Youan Hospital of Capital Medical University, Harbin Medical University, the Second People’s Hospital of Tianjin, the First Hospital of Changsha, the Eighth People’s Hospital of Guangzhou, Liuzhou General Hospital, the Third People’s Hospital of Guilin, the Third People’s Hospital of Shenzhen, Guiyang Public Health Clinical Center, Public Health Clinical Center of Chengdu, Kunming Third People’s Hospital, Yunnan Provincial Infectious Disease Hospital, the Fourth People’s Hospital of Nanning, Guangxi Longtan Hospital, the First Affiliated Hospital of Zhejiang University, and Xixi Hospital of Hangzhou. This protocol has been written in accordance with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) statement . The enrprogressive dyspnoea, fever and non-productive cough within days to weeks,diffuse “ground glass” interstitial infiltrates spreading from the hilum in a chest radiograph,2 gradient, (A-a)DO2 ≥ 35 mm Hg, or room air arterial oxygen, PO2 < 70 mm Hg.alveolar-arterial OThe presumptive diagnosis of moderate to severe PCP will have to meet the following criteria , 11:proPneumocystis cysts or trophozoites via staining or detection of Pneumocystis DNA via polymerase chain reaction in sputum samples, bronchoalveolar lavage fluid, or biopsy samples in addition to the criteria for the presumptive diagnosis of moderate to severe PCP. We define PCP-immune reconstitution inflammatory syndrome (PCP-IRIS) as occurring when the subject experiences a paradoxical exacerbation of either clinical symptoms or radiological signs of PCP after the initiation of ART despite receiving appropriate drug treatment for PCP.The definitive diagnosis of PCP requires identification of are at least 18 years old,have a confirmed diagnosis of HIV-1 infection,have a diagnosis of moderate to severe PCP presumptively or definitively,have not received any antiretroviral treatment,are willing to give informed consent.Subjects will be included in our study if they satisfy the following criteria:are allergic or intolerant to any of the prescribed therapeutic drugs;9/L, neutrophil count < 0.5 × 109/L, platelet count < 50 × 109/L, blood amylase > 2 × upper normal limit (UNL), serum creatinine > 1.5 × UNL, aspartate aminotransferase/alanine aminotransferase/alkaline phosphatase (AST/ALT/ALP) > 5 times of UNL, total bilirubin > 2 × UNL, serum creatine phosphokinase > 2 × UNL;have haemoglobin < 60 g/L, white blood cell count < 1.0 × 10have unstable concomitant OIs other than PCP;have serious heart disease, brain disease, lung disease, kidney disease, tumour disease or other systemic diseases;are pregnant or breastfeeding;have severe mental illness;are intravenous drug users;are not of Chinese nationality.Subjects will be excluded from the study if they:A specific random number sequence will be generated by the Medical Research Platform  for eachAll of the results will be recorded and double-entered independently. All data will be documented on case report forms and immediately recorded in the database through the Medical Research Platform. Missing values will be checked to ensure data completeness as much as possible. Data that are significantly abnormal or outside the clinically acceptable range  must be explained, and the necessary explanation must be given by the physician. Drop-outs and AEs will be recorded in time, and drugs used for the trial will be supplied, stored, distributed and recycled in accordance with relevant regulations. To ensure the high quality of this trial, the members of the quality management committee will diligently train all researchers involved in the trial in accordance with a standard operating procedure before the trial begins. In order to encourage the subjects to complete follow-up and improve their compliance, each subject will be appropriately counselled by health-care providers before treatment.Data collected during the course of the research will be kept strictly confidential and accessed only by members of the trial team. Each participant will be allocated an individual trial identification number, and details regarding participants will be stored on a secure database, which is protected by account number and password. No one other than the investigators in this study, the members of the ethical committees in each centre, and the members of the trial steering committee will have access rights to the data set. Anonymised trial data will be shared with other researchers to enable international prospective meta-analyses.During implementation of the trial, the data monitoring committee in the coordinating centre described above will monitor protocol deviations and the consistency of the database and the archives. After the trial is completed, a data management report meeting will be held to guarantee the validity and authenticity of this trial. At the same time, the data monitoring committee will evaluate the specific protocol deviations related to each subject and will exclude patients with major deviations from final analysis of the database. The members of the data monitoring committee are independent of the sponsor, and all members of the committee declare that they have no conflicts of interest. The data administrator will perform a database lock after the data lock record is signed by the principal investigator, sponsor, statistical analysts and data managers.This study is an open-label design, where both the investigator and the patient are aware of which assigned group the patient belongs to. The outcome will be independently assessed by three infectious disease physicians appointed as blinded outcome assessors, who will be provided with all study data available except for identification information and treatment allocation for the participant. If the three outcome assessors hold different opinions, the final assessment result will be subject to majority opinion. Data analysis will be performed by analysts in the School of Biomedical Engineering, Capital Medical University, Beijing, China. All analysts will process the data recorded in the Medical Research Platform  without All subjects will receive conventional treatment for PCP in accordance with the recommendations of Chinese guidelines for diagnosis and treatment of HIV/AIDS (2018) . TMP-SMZThe primary endpoint is incidence of disease progression at week 48. Disease progression is defined as any new or relapsing HIV-related OI, which has been listed in the Guidelines for the Prevention and Treatment of Opportunistic Infections in Adults and Adolescents with HIV , and deaThe secondary endpoints are the changes in CD4 counts from baseline to weeks 12, 24 and 48; virological suppression rate at weeks 24 and 48; rates of development of PCP-associated IRIS, and AEs over 48 weeks, including (1) grade 3 or 4 AEs which will be graded using the Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Paediatric Adverse Events (version 2.1) , (2) ser+ cells increase significantly from baseline; and (3) previously diagnosed OIs, newly diagnosed OIs, and drug toxicity are excluded.Virological suppression is defined as the HIV RNA level of a patient below the lower limits of detection of currently used assays (50 copies/mL). A subject would be considered to have a diagnosis of PCP-associated IRIS if he or she meets the following criteria : (1) detThe sample size calculation was conducted using PASS software version 15 . Previous studies have shown that the incidence of disease progression with deferred ART in patients with acute OIs was 24.1%. In the preceding study, 63% of patients with acute OIs were diagnosed with PCP . The undThe primary outcome analysis will be conducted using the intention-to-treat exposed (ITT-E) population, which consists of all randomly assigned patients, regardless of whether they are in full compliance with the study protocol. ITT-E will be used to assess the primary efficacy endpoints. We also plan to analyse the primary outcome using the per-protocol analysis set, which excludes subjects who do not follow the treatment regimens. If any data are not recorded, the multiple imputation method will be used. Baseline will be defined as the date of randomisation.For the primary endpoint, we will compare the incidence of disease progression at week 48 between the two groups using the chi-squared test. Supportive statistical analysis will include time-to-event methods with the Cox proportional hazards model to include baseline CD4 counts, HIV regimens, and antifungal drug use. t tests. To explain the competing risk of death, the cumulative incidence function will be used to compare AEs and IRIS between the two groups [P value of less than 0.05 will be considered statistically significant. All analyses will be performed using SPSS Statistics version 24.0 . It is possible that there will be an interim data evaluation. If there are interim looks at the data, a serial gate-keeping procedure will be proposed to control type I error resulting from multiple interim analyses.For secondary endpoints, categorical variables, including proportion of virological suppression, incidence of PCP-related IRIS, and incidence of AEs, will be analysed using Fisher’s exact test. Continuous variables, including CD4 count change, and other potential outcome parameters will be analysed by Wilcoxon tests or o groups . A P valThe trial steering committee, consisting of the primary investigator of the entire project, Yaokai Chen, and the Administration Office for National Science and Technology Major Projects, provides overall supervision and ensures that the trial is properly registered. The primary investigator oversees the evolution of the trial every two months and meets with representatives of the Administration Office for National Science and Technology Major Projects once or twice per year. After the trial is completed, the Administration Office for National Science and Technology Major Projects will evaluate the quality of the trial. The Clinical Research Center of Chongqing Public Health Medical Center, which serves as the coordinating centre of the trial, is responsible for the coordination and monitoring of all 17 research sites. The coordinating centre will track trial progress at least once a month by networking, phone call, email or on-site monitoring. The 17 sub-primary investigators, one for each site, are responsible for coordinating and organizing the enrolment, informed consent, and follow-up procedures at their local sites. There is no stakeholder and public involvement group in this trial.ChiCTR1900021195). We will share the results through published medical journal articles and a conference presentation after completion of the study.The study was approved by the ethics committee of the Chongqing Public Health Medical Center (2019–003-02-KY) and duly registered at the Chinese Clinical Trial Registry  found that patients with AIDS-defining diseases, including those with PCP, were significantly more likely to progress to a new AIDS-defining disease or death if ART initiation was deferred more than 30 days after HIV infection diagnosis in comparison with early ART initiation patients (<30 days after HIV infection diagnosis) . In 2009Herein, we designed a multi-centre prospective randomised controlled trial in China, in which all eligible subjects will be randomly assigned to an early ART initiation group (≤14 days after PCP diagnosis) or a deferred ART initiation group (>14 days after PCP diagnosis). We will collect data of survival, immunological reconstitution, virological suppression, AEs, and IRIS emergence in HIV-infected patients with moderate to severe PCP, and the aim is to investigate the safety and benefits of early ART. We speculate that subjects in the early ART initiation group will have a lower incidence of disease progression than those in the deferred ART group. We hope that our results will provide unequivocal clinical evidence as to the optimal timing to initiate ART in HIV-infected patients with moderate to severe PCP.This trial is in the recruitment phase. Patient recruitment began in March 2019 and was expected to be completed in May 2020 .Additional file 1.


Older age, higher baseline eGFR, higher VCY and longer exposure to INI treatment were associated with eGFR reduction at univariate analysis. In the multivariate model, older age (p = 0.039), baseline eGFR (p<0.001) and VCY (p = 0.069), were retained. We also observed a longer exposure to PI/b and INI in patients with lower control on HIV-RNA, expressed as VCY>2 log10. Our study outlines a progressive eGFR reduction in young adults with PHIV, related to the lower control on HIV-RNA VCY and related to aging.Among people with perinatal HIV infection (PHIV), non-communicable diseases, such as chronic kidney disease, are increasing. Both HIV replication and antiretroviral therapy are recognised causes of renal impairment. Objective of the study is to describe the impact of viremia copy-years (VCY) and antiretroviral therapy on trend of estimated glomerular filtration rate (eGFR) in a cohort of adults with perinatal HIV infection. We conducted a multicentre observational study in sixty adults living with PHIV across a 9-year period, from January 2010 to December 2018. The mean values of eGFR were analysed at the first (T0) and last year of observation (T1). VCY was defined as the area under HIV-RNA curve during the study period. We analysed data according to antiretroviral therapy: tenofovir disoproxil (TDF), non-nucleoside reverse transcriptase inhibitors (NNRTI), boosted protease inhibitors (PI/b), integrase inhibitors (INI). We observed a mean overall eGFR reduction from 126.6 mL/min (95%CI: 119.6–133.5) to 105.0 mL/min (95%CI: 99.55–110.6) ( Since the early 2000s, there has been a gradual reduction of perinatal HIV infection (PHIV), owing to the extensive use of modern antiretroviral therapy (ART) in pregnant women and prophylaxis in newborns , 2. Parawww.reteligureHIV.it)–an online database for anonymous and automatic data collection—was used to retrieve clinical data in Genoa ). In the 10. Patients with higher VCY values had longer PI/b and INI exposure. This can perhaps be attributed to the higher frequency of resistance mutations in viral genotype, common among patients with PHIV exposed to older, suboptimal ART regimens for decades [10. Data on the effect of VCY on eGFR trend are lacking in the literature. However, a greater VCY has been correlated with hypertension and higher mortality [p = 0.069). A previous clinical diagnosis of AIDS, a lower CD4+ nadir, drug abuse or current smoking were not related, in our analysis, to eGFR reduction. Our data suggests that monitoring renal function in people with PHIV is mandatory despite the ART regimen prescribed, and that maintaining viral suppression could be a determinant factor in preserving renal function in patients with PHIV. Limitations of this study are its retrospective nature that restricts us from evaluating the influence of possible unmeasured confounders on the eGFR trend, the absence of a control group that prevents us from comparing the worsening trend observed among the studied PHIV patients with a healthy population and the absence of other laboratory markers of renal damage.In this analysis, we offer new data about cumulative VCY and eGFR trend among people with PHIV. Our analysis is one of the first describing eGFR trend in adult patients with PHIV. This trend is expected to be worse compared to that of a healthy population, where factors of kidney damage such as ART exposure and HIV infection are absent , 9. In p decades . Further decades , 27–29. ortality . We suggOur study outlines a progressive eGFR reduction among young adult patients with perinatal HIV infection, related to higher VCY and to aging. More efforts are needed in monitoring renal function and maintain stable viral suppression in patients with PHIV.


All classes of antiretroviral therapy (ART) have been implicated to induce adverse drug reactions such drug‐induced liver injury (DILI) and immune‐mediated adverse reactions in Human Immunodeficiency Virus (HIV) infected individuals. Patients that develop adverse drug reactions tend to have prolonged stays in hospital and may require to change to alternative regimens if reactions persist upon rechallenge or if rechallenge is contraindicated due to severity of the adverse reaction. Diagnosis of DILI remains a huge obstacle that delays timely interventions, since it is still based largely on exclusion of other causes. There is an urgent need to develop robust diagnostic and predictive biomarkers that could be used alongside the available tools  to give a specific diagnosis of DILI. Crucial to this is also achieving consensus in the definition of DILI so that robust studies can be undertaken. Importantly, it is crucial that we gain deeper insights into the mechanism of DILI so that patients can receive appropriate management. In general, it has been demonstrated that the mechanism of ART‐induced liver injury is driven by four main mechanisms: mitochondrial toxicity, metabolic host‐mediated injury, immune reconstitution, and hypersensitivity reactions. The focus of this review is to discuss the type and phenotypes of DILI that are caused by the first line ART regimens. Furthermore, we will summarize recent studies that have elucidated the cellular and molecular mechanisms of DILI both in vivo and in vitro. The disease was estimated to affect 37.9 million people worldwide in 2018, directly causing approximately 770 000 deaths.The successful roll out of antiretroviral therapy (ART) has reduced the risk of early mortality due to opportunistic infections and improved both the prognosis as well as the quality of life for HIV‐infected patients. There was an estimated 24.5 million people receiving ART worldwide in 2018, a significant increase from 7.7 million people that were accessing ART in 2010.Unfortunately, some patients receiving ART may develop severe adverse drug reactions such as drug‐induced liver injury (DILI). The reported incidence of ART‐induced liver injury varies greatly due a number of factors such as a criteria used to define the severity of hepatotoxicity, geographical location, prevalence of chronic viral hepatitis infections, genetic heterogeneity in populations, frequency of liver enzyme determinations, medication prescribing patterns, and other exogenous exposures.In this review, we will discuss the different patterns and severity of DILI and describe the types and phenotypes of DILI caused by ART in HIV‐infected individuals. We will also provide a summary of the studies that have analysed the molecular mechanisms of DILI with a particular focus on cellular toxicity, immunological responses, and biochemical pathways that have been implicated to drive hepatotoxicity of first line ART. It is important to note that the WHO recently introduced new guidelines that recommended the use of dolutegravir alongside two nucleoside reverse transcriptase inhibitors (NRTIs) as a first line regimen.1.1R value  ÷ ). DILI is categorized as hepatocellular when R is more than 5 (R > 5), cholestatic when R is less than 2 (R < 2), and mixed when R is more than 2 but less than 5 (2 < R < 5).There has been great variability in the criteria used in clinical studies to define DILI and/or the severity of hepatotoxicity. Some studies have defined DILI as elevations in liver enzymes  two times the upper limit of normal (ULN),The DILI Expert Working Group and AIDS Clinical Trial Group graded DILI severity into four grades based on baseline ALT or alkaline phosphatase (ALP) below the ULN.+ cell count that was greater than 200 cells/mm3.All classes of ARTs cause DILI in HIV‐infected patients; however, some classes are more toxic than others. Table Nucleoside reverse transcriptase inhibitors, protease inhibitors (PIs), and fusion inhibitors have also been reported to cause DILI in HIV‐infected patients. NRTIs such as zidovudine, stavudine, and didanosine caused moderate to severe DILI while emtricitabine, abacavir, and tenofovir induce minor elevations in liver enzymes.1.2Drug induced liver injury (DILI) has been mainly classified as either direct or idiosyncratic; however, indirect injury is emerging as a third class.1.2.1Clinically, the most common feature of direct hepatotoxicity is acute hepatic necrosis, and severe cases present with acute hepatic failure characterized by encephalopathy and coagulopathy.1.2.2R value; hepatocellular, cholestatic, and mixed.Idiosyncratic hepatotoxicity occurs in rare cases  and is usually caused by drugs with little or no intrinsic toxicity to the liver.1.2.3Indirect hepatotoxicity is caused by the action of the drug in triggering a new liver condition or worsening an underlying condition such as viral hepatitis than its intrinsic toxicity or idiosyncratic properties.1.31.3.1CYP2B6 slow metabolizer genotype with NVP driven hypersensitivity reactions (rash) but not with hepatotoxicity.CYP2B6 slow metabolizer genotype was associated with an increased risk of EFV adverse reaction.CYP3A4 rs4646437 genotype.CYP2B6 haplotype *6/*6 was identified in 8.2% of the HIV‐infected patients that received an EFV‐based regimen and it was associated with high ALP and total bilirubin, suggesting that this genotype may correlate with an increased risk of hepatotoxicity in patients taking an EFV‐based ART regimen.CYP2B6 *6/*6 genotype with ART‐induced liver injury and high plasma concentrations of EFV.CYP2B6 genotypes so they could be given therapy that would have minimal adverse effects to encourage compliance treatment or know the most likely CYP2B6 genotypes within a population to decide on pragmatic therapy.Genetic variations in drug metabolizing enzymes have been associated with risk of adverse drug reaction in patients taking ART Table . A study1.3.2IL‐1RN (VNTR) and IL‐1β (‐511C/T) polymorphisms in 34 HIV‐positive patients with ARV hepatotoxicity, 128 HIV‐positive patients without hepatotoxicity, and 152 healthy controls using PCR and PCR‐RFLP. They found that IL‐1RN 2/2 and 1/3 genotypes were highly represented in patients with hepatotoxicity to a NVP based ARV regimen.IL‐1β‐511CT and ‐511TT genotypes enhanced the risk of hepatotoxicity in patients taking the NVP based regimen. Finally, a study by Asensi and colleagues showed that IL‐1β3954T allele is associated with lipodystrophy syndrome in patients on ART.The immune response plays a critical role in driving some of the adverse drug reactions that are associated with ART in HIV‐infected individuals. The severity of DILI is influenced by the presence of toxic molecules and a critical counterbalance of cytokines such as tumor necrosis factor‐alpha, interleukin‐1beta (IL‐1β), IL‐10 and IL‐1RN.1.3.3The interactions between the T cells and professional antigen presenting cells via the class I or class II human leucocyte antigen (HLA) plays a key role in development of adverse drug reactions. In fact, HLA‐B*5701 polymorphism has been linked with abacavir hypersensitivity in HIV‐positive individuals,1.41.4.1The molecular mechanism of hepatotoxicity induced by ART seems to be multifactorial. A number of studies have reported that EFV causes cellular damage indicated by changes in the cells morphology, endoplasmic reticulum (ER), and mitochondrial integrity.Mitochondrial toxicity is a common pathway linked with the use of NRTIs in HIV positive individuals.1.4.2Research has implicated the involvement of respiratory chain proteins,sirt3 gene encoding Sirtuin‐3, a major mitochondrial NAD+‐dependent deacetylase in mice (Sirt3 knockout mice) protected hepatocytes from EFV‐induced cytotoxicity compared to cells from wild‐type mice.Efavirenz is known to increase the activity of AMPK, which results in increased production of ROS and possibly initiates cell death.Another interesting biochemical family of serine/threonine kinase is the JNK. These are important regulators of cellular stress responses including modulation of cell death.A study by Lee et al showed that in murine hepatocytes, the high concentrations of isoniazid alone did not cause acute cell injury.1.4.3Careful analysis of cellular morphology by transmission electron microscopy (TEM) revealed autophagic degradation of the mitochondria and the ER appeared to be wrapped around the mitochondria, possibly in order to generate a membrane that would later be incorporated into the autophagic vacuoles.At cellular level, treating Hep3B cells with lower concentrations of EFV has been shown to induce autophagy as evidenced by the presence of autophagic vacuoles and expression of specific autophagic protein markers such as microtubule‐associated protein 1A/1B light chain 3 and Beclin‐1.2Adverse drug reactions including DILI present an enormous challenge for the treatment and management of HIV‐infected patients. This is further compounded by other complexities such as the emergence of drug resistance, introduction of new multidrug regimens, and co‐morbidities like TB and viral hepatitis. One cannot underscore a need for a breakthrough in diagnosis of DILI and how this will aid in the management of the adverse reactions so that patients can adhere to their treatment regimen. A good example to cite here is the success that has been achieved by taking a precision medicine approach in dealing with the abacavir hypersensitivity and implementing testing for HLA‐B57:01 genotype prior to prescribing the treatment.CYPs) have been associated with hepatotoxicity and hypersensitivity reactions to some ART.Efforts have been made over the years to achieve consensus in the definition of DILI.The immune system plays a critical role in augmenting and mediating some of the adverse reactions such as DILI and hypersensitivity reactions. Studies have incriminated polymorphisms in cytokine genesThe authors have no conflict of interest to declare.JP, MJM, NK, WCS, and HN: conception and design of manuscript; JP and HN: manuscript writing; JP, MJM, NK, WCS,and HN: reviewed and edited the manuscript; HN and MJM: provided financial and administrative support; HN: final approval of the manuscript.


A strategy implemented by the South African Department of Health to manage the high burden of human immunodeficiency virus (HIV) has been to task-shift services to primary health care clinics. Outcomes of paediatric patients with HIV are poorer than those of adults, particularly in rural areas. Viral suppression in paediatric patients at the feeder clinics of a rural South African hospital was anecdotally far below the aim of the Joint United Nations Programme on HIV/AIDS (UNAIDS) of 90%.A quality improvement approach was used to conduct a baseline assessment of HIV viral suppression in paediatric patients and other process measures, implement a clinical mentorship intervention and evaluate its effectiveness.An initial audit of 235 clinical folders of paediatric patients with HIV revealed a viral suppression of 55.3%. Other poor measures included prescription accuracy, viral loads performed within schedule and response to successive high viral loads. A clinical mentorship intervention using dedicated doctor outreach was implemented and the audit repeated after 12 months (263 folders). Viral suppression improved to 67.4%, as did most other process measures.The quality improvement approach regarding the aim to significantly improve viral suppression in paediatric patients through the implementation of clinical mentorship was successful. As of early 2016, unreliable TIER.Net3 data (because of a lack of trained data capturers at clinics) put the viral suppression in paediatric patients at roughly 50% for the Madwaleni feeder clinics.Madwaleni Hospital is a deeply rural district hospital on the ex-Transkei coast of the Eastern Cape, South Africa. The concern of clinicians about the number of paediatric admissions with virally unsuppressed human immunodeficiency virus (HIV) pointed to poor viral suppression rates at its primary health care (PHC) feeder clinics. Paediatric HIV care was previously hospital-based and managed by a local non-governmental organisation (NGO).4 With a view to end this epidemic by 2030, UNAIDS recommended the implementation of the ‘90-90-90’ targets: 90% of the population tested for HIV, 90% of those who are HIV positive on antiretroviral therapy (ART) and 90% viral suppression for those on ART.5 In an attempt to increase access to HIV care, the South African National Department of Health implemented the strategy of task-shifting, which moved the initiation of patients on ART and their subsequent management to nurse-run primary health clinics.6 Studies have subsequently found nurse-monitored HIV care of adult patients to be non-inferior to doctor-monitored HIV care in the South African context.8According to UNAIDS data from 2018, 90% of the estimated 1.8 million HIV-positive children live in sub-Saharan Africa.9 A 2016 meta-analysis showed levels of viral suppression in paediatric patients in resource-limited settings were 73%,10 compared with 84% in adults,11 with a 2019 study from Eswatini showing 78% viral suppression in paediatric patients.12 Rural children are particularly vulnerable with studies showing increased risk for poorer outcomes compared with urban counterparts,14 and poorer viral suppression with a 2017 study conducted in rural Limpopo showing between 48% and 52% viral suppression.15Despite these global efforts, paediatric HIV care has not seen the same levels of success as adult HIV care.16 It has the advantage of incorporating both research and action in the same process.18 Support for and training in QI methodology were received from the Stellenbosch University Collaborative Capacity Enhancement through Engagement with Districts (SUCCEED).19To address the perceived poor HIV viral suppression in paediatric patients at the Madwaleni feeder clinics, a quality improvement (QI) approach was selected. The QI approach views health systems as a process with constant opportunity for improvement.The aim of this QI-driven research was therefore to improve the HIV viral suppression in paediatric patients at the Madwaleni PHC clinics from a presumed baseline of 50% to 90% within the space of 1 year (May 2016 to May 2017).20 came into effect from April 2015 and were used as the official guidelines for the duration of the QI cycle.Madwaleni Hospital and its nine PHC clinics are in the Mbhashe subdistrict of the deeply rural Eastern Cape. According to TIER.Net data, an estimated 230 children were in care and on ART at the PHC clinics in 2016. All nine clinics had at least one professional nurse (PN) who had undergone the Nurse Initiated Management of Antiretroviral Treatment (NIMART) training. The national consolidated guidelines for the prevention of mother-to-child transmission of HIV (PMTCT) and the management of HIV in children, adolescents and adultsAfter using the data from TIER.Net to identify the problem of poor viral suppression in paediatric patients, a research or QI team was convened consisting of key stakeholders, including the acting clinical manager, acting CEO, family physician and family medicine registrar from Madwaleni Hospital, the operational managers of the PHC clinics, as well as a volunteer social worker and volunteer clinical researcher medical officer. The baseline file audit was planned for May 2016 with a gold standard of 90% viral suppression set, and various process measures discussed and decided upon: viral load (VL) performed within schedule, months after second high VL before regimen change, number of efavirenz (EFV) to lopinavir or ritonavir (LPV/r) changes, occurrence of stavudine (d4T) prescriptions, number of weights in last 12 months, correct dosing according to last recorded weight and daily versus twice-daily dosing of ART where applicable.The baseline file audit date was set for 1st of May 2016. Data collection was performed using an Excel spreadsheet audit tool designed by the team. Inclusion criteria were all files where the child was less than the age of 15 years at the audit date and were considered ‘in care’, that is they had received ART at the PHC clinic within the 3 months preceding the audit date. No direct contact was made with the population for the purpose of data collection, and all data were collected from patient records only. To ensure confidentiality, patient names were removed during the data collection process and not recorded on the final data collection tool.Although this was a quality-improvement initiative, ethical clearance was still obtained from Human Research Committee, Faculty of Health Sciences postgraduate education, training Research and Ethics Unit, Walter Sisulu University (Clearance number: 033/2017). Site approval from the Madwaleni Hospital chief executive officer and provincial approval from the Eastern Cape Health Research Committee were also obtained.At the time of the baseline audit, 235 children were on ART and in care. With regard to the current ART regimens shown in 20 Only 18.1% of eligible patients were placed on daily ABC or 3TC dosing instead of twice-daily dosing. Daily dosing of ABC or 3TC is recommended by the national guidelines because of strong carer preference and a non-inferiority to twice-daily dosing in terms of viral suppression.2120 There was an average of 9.2 months’ wait before a repeat VL testing after a VL of more than 1000 RNA copies/mL, whereas the national guideline recommended a repeat after 2 months.20 Of the 219 eligible patients, only 121 (55.3%) had a suppressed VL. The definition of viral suppression used was a VL of less than 1000 RNA copies/mL performed within the last 13 months or performed within the last 7 months of ART initiation or a regimen change. In calculating viral suppression, patients who did not have their VL tested in the prescribed period were considered as having an unsuppressed VL. Viral suppression was slightly higher than the TIER.Net data of 50%, but still far below the UNAIDS goal of 90-90-90.As can be seen in 22 In considering the cause of low viral suppression in paediatric patients, and looking at solutions that worked in similar settings, a clinical mentorship initiative was decided on. This was made possible by senior stability and an increased number of doctors employed at Madwaleni hospital from 2013.The QI team made use of a combination of systematic team problem solving and benchmarking to develop an intervention to improve viral suppression in paediatric patients within the QI approach.23 A Botswana study looking at clinical mentorship for decentralised paediatric HIV care found that it may assist improvements in a number of important areas, including proper ART dosing and monitoring,24 whilst a QI study carried out in the North West Province of South Africa concluded that ‘task shifting in a paediatric ART programme in particular can be effective if patients continue to be closely monitored and PHC nurses are regularly supported by a PHC doctor’.25A study in Khayelitsha (South Africa) had found that as a result of clinical mentorship ‘nurses improved their confidence in performing HIV related clinical tasks, nurses were initiating patients after mentorship and the quality of initiation and management was satisfactory’.26 The purpose of the doctor outreach was to assist by seeing complicated patients  together with the PNs. This was not limited to HIV-positive patients. Ongoing nurse training or education in the form of short sessions around different clinical topics were conducted on an ad hoc basis to strengthen the problem solving and clinical decision-making of the nurses,27 whilst increasing their confidence in managing ART.23 In addition to this, the doctors were asked to review a few folders having records of paediatric patients with HIV at each visit to monitor services and data.28From May 2016 a medical officer, community service medical officer or family medicine registrar was assigned a specific clinic which he or she visited on a monthly or fortnightly basis, depending on the total number of patients seen at the clinic per month. As far as possible, the same doctor did outreach to the same clinic, to promote continuity of care and assist in developing an effective clinical mentorship relationship with the clinic staff.After 12 months, the audit was repeated using the same audit tool with the same inclusion criteria with a re-audit date of 1st May 2017. Data were captured and analysed using Microsoft Excel. Chi-squared test or Fisher exact test were used to test for association between categorical variables. Continuous data were tested for normality, and the appropriate statistical test was used to compare for significance of association between groups.As can be seen in p < 0.0001). The number of patients changing from an EFV-based regimen to an LPV/r-based regimen had also increased from 1.6% to 8.3% (p = 0.021), implying that more patients were being changed from failing first-line regimens to second-line regimens.Conversely, statistically significant differences can be seen in 20 dropped from 51.1% to 35% (p = 0.0004) – whilst underdosing almost halved from 11.7% to 6.4% (p < 0.0001) from 2016 to 2017. The percentage of patients being prescribed a daily instead of twice-daily dosing of eligible ART also increased significantly from 18.1% to 44.2% (p < 0.0001).The frequency of weights documented did not differ significantly from 2016 to 2017, but there was a statistically significant reduction in prescription errors as shown in p < 0.0001) of patients had their VL taken within schedule in 2017 compared with 2016. The average wait for a repeat VL testing after an initial VL of more than 1000 RNA copies/mL also decreased from 9.2 to 5.3 months (p = 0.0011). With regard to the primary aim of the study, the percentage of suppressed patients in 2017 also significantly increased from 55.3% to 67.4% (p = 0.0088).As shown in The use of a QI approach with a clinical mentorship intervention has resulted in significant improvements to various process measures, including an overall improvement in viral suppression in paediatric patients.29 The number of patients changed from an EFV-based regimen to an LPV/r-based regimen increased, implying that more children were changed from a failing first-line regimen with a non-nucleoside reverse transcriptase inhibitors backbone to a second-line regimen with a protease inhibitor backbone. At baseline, more than half of patients had an incorrect dose prescribed in the last year, which improved to 35% at the second audit. Although this remains alarmingly high, it is in keeping with findings from a study in a similar rural setting30 and could be an indication of the complexity of paediatric prescribing.31 Underdosing significantly improved from 11.7% to 6.4%, whilst the number of daily dosing prescriptions for ABC/3TC combination more than doubled from 18.1% to 44.2%. Viral load monitoring and response to VLs improved, with 16% more patients having their VLs performed within schedule, and the average delay between repeating a VL testing after an initial high VL down from 9.2 to 5.3 months, which is still longer than the 2 months recommended by the national guidelines.20 The most significant of the clinical findings was an improvement in viral suppression from 55.3% to 67.4%. Despite the improvement, this is still far short of the 90% suppression goal.There was a dramatic reduction in the amount of d4t prescriptions in accordance with the 2013 recommendation to switch children from d4t-containing regimens owing to its potentially severe metabolic toxicities.A significant limitation of the study was the 12 months allowed for the intervention before its effect was assessed. We anticipate that repeated data collection after a further 12 months would have shown further improvement in viral suppression. This is due to the fact that there is a minimum of 8 months from when the initial high viral load of a failing first line regimen is taken, to when the initial viral load on a second line regimen is taken. A further limitation was the lack of standardisation across the clinics with regard to the implementation of the clinical mentorship. Confidence in dealing with paediatric HIV varied from doctor to doctor, as did enthusiasm for PHC. This led some doctors to taking more initiative than others. A general limitation of the QI process is that we are unable to prove causation. During 2016 and 2017, more data capturers were employed at the clinics, which could also have contributed to improved outcomes.Recommendations for future QI cycles include interventions focussing on data management, specifically data capturers and their interaction or communication with clinicians. The TIER.Net system has functions that enable the flagging of patients where VLs are due or where consecutive VLs are more than 1000 RNA copies/mL. We hope to improve the use of TIER.Net, its data accuracy and the use of its functions. The employment of extra data capturers at the clinics during 2017 has made this a feasible option. Additional recommendations include interventions focussing on prescription error rates, specifically improved dosing by weight. One suggestion has been to develop a weight-based ART dosing wheel .The QI approach to addressing poor viral suppression has proved to be beneficial at the Madwaleni feeder clinics. We anticipate that this positive exposure to QI will encourage the Madwaleni doctors and clinic nurses alike to make use of this approach in the future.Twelve months of clinical mentorship appears to have had a positive impact on viral suppression and paediatric HIV care in general. The ongoing doctor support of PHC nurses in fulfilling their task-shifting role in this very rural context appears to be crucial to the programme’s success.