Uganda adapted Viral load (VL) testing for monitoring HIV treatment success and virologic failure. However, there is a paucity of data on how the VL testing guidelines are followed in practice in the HIV clinics. This study determined the adherence to national guidelines on VL testing, barriers, and associated factors in persons living with HIV (PLHIV) on ART in southwestern Uganda. We conducted a cross-sectional mixed methods study from April to May 2021 at four HIV clinics in southwestern Uganda. Patient chart review using a checklist that captured age, gender, and level of a healthcare facility, dates of ART initiation, dates VL specimens were drawn, line of ART, patient adherence to ART was done. Continuous data were summarized using mean and median and Chi-square was used for categorical data. We performed regression analysis to determine factors associated with adherence to viral load testing guidelines at a 95% level of significance. Key informant interviews with managers of the health facility, ART clinic and laboratory were carried out, and thematic analysis was conducted to explore barriers to adherence to VL testing guidelines.p = 0.002) and increasing patient age  were the factors associated with adhering to VL testing guidelines. Long turnaround time of VL results and insufficient VL testing kits were cites by providers as barriers.The participants’ mean (SD) age was 39.9(± 13.1) years, 39.5% were male, 45.8% received care at a general hospital and median duration on ART was 5 years . Of the 395 patient charts reviewed, 317 had their VL testing (80.3%) per the guidelines (defined as up to one month post due date). Receiving care at a hospital (aOR = 2.20; 95%CI 1.30–3.70; We found suboptimal adherence to VL testing guidelines in PLHIV on ART in southwestern Uganda. Increasing patient age and getting care at a higher-level health facility were associated with guideline-based viral VL testing. Long turnaround time of VL test results and inadequate test kits hindered compliance to VL monitoring guidelines. Strategies that target young PLHIV and lower-level health facilities, increase the stock of consumables and shorten VL results turnaround time are needed to improve adherence to VL testing guidelines. In an effort to end the human immunodeficiency virus (HIV) pandemic, the World Health Organization (WHO) recommended the initiation of antiretroviral therapy (ART) for all people living with HIV (PLHIV) regardless of their clinical and immunological status  and the nd and 3rd line ART regimen options [There is evidence that regular VL monitoring is a cost effective  and is t options .Delays in VL testing are likely to be related to patient, provider, and system-level factors . PatientThis study aimed at finding out the extent to which the national guidelines on VL testing in PLHIV were being implemented at healthcare facilities in southwestern Uganda. The factors associated with guideline-based VL testing, provider perceived barriers to VL testing at the same health facilities were also of interest.We conducted a concurrent explanatory mixed methods cross-sectional study using secondary data as a quantitative component and a descriptive qualitative component. The study was conducted as a single snapshot between April and May 2021. However, we abstracted data spanning the period from February 2018 to February 2020 prior to the first COVID-19 lockdown in Uganda which was characterized by significant disruption in patient visit schedules , 17.We conducted the study at four rural HIV clinics in southwestern Uganda. The estimated region’s population is 5 million people . Uganda’Health facility records show that in 2019, the number PLHIV on ART in the HIV clinic were 2,447 at Kitagata hospital, 1,908 at Itojo hospital, 1,300 at Kinoni HCIV and 2,131 at Kabwohe HCIV. The HIV clinic’s clinical care team usually consists of a doctor or clinician who serves as the manager, trained nurses and midwives, laboratory technicians, counsellor, records/data clerk and PLHIV peers. Every PLHIV has a unique clinic identifier assigned at the health center. His/her sociodemographic and clinic data from the treatment card (blue card) are entered into health facility’s electronic medical records (EMR) database. The PLHIV routinely have one clinic visit every 3 months for clinical assessment and drug refills. When the scheduled visit is for VL testing, the date of blood sample collection is documented on the client’s blue card. The drawn VL samples are sent from all HIV clinics to a district laboratory hub for transportation via the National Sample and Results Transport Network to the Central Public Health Laboratories (CPHL) that conducts countrywide VL testing. VL results are returned to the facility and recorded in viral load registers and patient charts (blue cards) .For the quantitative component, we focused on medical records of PLHIV on ART during the study period, between February 2018 and February 2020. Eligible individuals needed to have been on follow-up care in the same ART clinic for at least 6 months after initiating the ART. For the qualitative component, we interviewed health facility, laboratory, and ART clinic mangers.th ID until 105 participant IDs were generated.For the quantitative component, a sample size of 420 participants was estimated using the Kish Leslie formula for a single population proportion . We consFor the qualitative component, we purposively sampled 12 providers by selecting the three focal persons per health facility for the four participating sites, i.e. health facility, HIV clinic and laboratory manager.This was a mixed method study which applied both quantitative approaches for patient data and qualitative approaches for healthcare providers data. The database used in this study was derived from PLHIV medical records and contained detailed information about demographic and HIV care characteristics. For each PLHIV’s ID selected, required data was extracted from blue card by trained research assistants using the checklist which was designed based on the details captured by the Uganda laboratory requisition forms. The checklist used abstracted age, sex, and level of healthcare facility, dates of ART initiation, dates VL specimens were drawn, line of ART, patient adherence to ART. The ART start date was used to calculate the duration on ART at the time of the VL test.For the qualitative component, the research assistants trained in qualitative research used an interview guide  designed and pretested based on the predetermined research outcomes to conduct key informant interviews with a purposively selected health facility manager, laboratory lead and ART clinic lead at each study site. Written informed consent for key informants was obtained prior to conducting the interviews which lasted between 40–60 min. Data was recorded using audio recorders and then backed up on an eternal hard drive.For the quantitative component, the outcome variable was adherence to VL testing guideline defined within one month from the due date. The independent variables were the PLHIV’s age, sex, duration on ART, ART adherence, ART line, VL test indication (routine versus suspected failure), previous VL result and health facility level.For the qualitative arm, outcomes of interest were emerging themes on barriers to adherence to VL testing guidelines as perceived by healthcare providers.www.epidata.dk, version 7.2.1) database and imported into STATA  version 15.0 for analysis. Descriptive analysis was conducted on the socio-demographic variables of age, sex, and level of the facility. The extent of adherence to the national VL testing guidelines was calculated as a proportion of total study participants who received VL testing on schedule. Univariable logistic regression was used to determine unadjusted association between sociodemographic, clinical and laboratory variables with the guideline-based viral load testing. All independent variables with p < 0.2 were then entered into a multivariable logistic regression model to determine factors independently associated with guideline-based viral load testing. Odds ratios (OR) and 95% confidence intervals (CI) were calculated and p-values < 0.05 considered statistically significant in all cases.For quality assurance, research assistants were trained on the essential study documents, data collected with the checklist by the research assistants was reviewed by the supervising investigator and any errors found were rectified. The qualitative interviews were conducted by experienced research assistants. Completed questionnaires were entered into an EPI-Info ; Protocol reference number: 11/02–21. A waiver of consent was obtained to access the required patient records and abstract research data. Written informed consent was obtained from all qualitative study participants.Of the 420 charts retrieved, 25 charts had missing data on date of VL testing or ART start and were excluded from analysis. Of the 395 patient charts with complete data, 214 were from HCIV (54.2%) and 181 were from general hospital (45.8%). Majority of the participants were female 60.5% The mean (SD) age was 39.9(13.1) years, and median (IQR) duration on ART was 5(3–7) years as shown in TableThe VL testing guidelines were adhered to among 317 of the 395 participants, equivalent to 80.3 per cent (95%CI:76.0–83.9), defined up to one month post due date.p = 0.002) and increasing patient age , as shown in Table Among the PLHIV, the factors associated with guideline-based VL testing were receiving care at a hospital  have not come. And they(patients) have to wait for subsequent visit find out what were their results”. .“Like now, we just received results of January to February (2021). And they are not for all the orders we made, others are missing or will take months before CPHL resends.” (Manger HIV clinic).“Like you bleed a client, this is April and you expect the results to come back in May but the results come back in June or July.” , online and phone calls were found to be major sources of confusion at the onsite labs leading to delays and misplacing of patient results”. Manager HIV Clinic.“Previously, it was inadequate sample collected. But that has since reduced and it is now issues of mismatching the request form with the samples. Sometimes you find a sample is collected, and then the form reaches the CPHL before the sample. So, you find it is rejected from that side”. (Manager laboratory).Also, the providers felt that unpredictable frequent switch between electronic and printed request forms was a barrier to VL testing.“At times, we get out of stock of kits, viral load kits, for collecting the samples”. (Manager laboratory).“You can order for a test, and you find that the viral load kits are not in the laboratory.” (HIV Clinic In- charge).Furthermore, providers felt that inadequate stocks of VL testing kits was hindering VL testing.In this mixed-methods cross-sectional study of healthcare providers and PLHIV on ART at HIV clinics in southwestern Uganda, we found suboptimal adherence to VL testing with eight in ten eligible persons being tested per guidelines. Increasing patient’s age and receiving care at the hospital were associated with adherence to VL testing guidelines. Inadequate VL sample collection kits, delay in return of VL results and ambiguities in laboratory request form hindered adherence to VL testing guidelines.We found lower adherence to VL testing than the 95% target . HoweverWe found a significant increase in adherence to VL testing guidelines with increasing age of the PLHIV. This agrees with findings of studies in Uganda  and SoutIn addition, this study found that PLHIV who received HIV care at hospital were twice likely to receive VL testing as per the guidelines when compared those in care at HCIV. This was consistent with studies done in Zimbabwe  and UganHealthcare workers perceived inadequate VL testing consumables and long turnaround time of VL test results as barriers to adherence to guidelines. Optimally, turnaround time should not exceed weeks from sample draw . SimilarMore broadly, our study shows the challenges to VL implementation in resource-poor settings associated with poor adherence to the national VL testing guidelines. It is possible that providers may not conduct VL testing on patients who attend their scheduled visits depending on their perception of available consumables. To optimize the VL testing cascade and improving compliance, and achieve the UNAIDS 2030 last “95”, these gaps must be addressed.The strengths of our mixed-methods cross-sectional study include random sampling of patient charts and exploring the provider perceptions that reflect the real-world situation of viral load monitoring in rural Uganda. Our study has some limitations. We retrospectively analyzed secondary programmatic data with notable missingness for data on VL testing dates (6%). Nevertheless, this did not influence study findings. Also, the study was conducted in one region of Uganda which limits generalizability. But, we believe the findings can be extrapolated to other low-income and rural settings.We found that adherence to VL testing guidelines was achieved for eighty percent of PLHIV attending HIV clinics in southwestern Uganda. Adherence to VL testing guidelines improved with increasing patient age and receiving HIV care at hospital-level health facility. Adherence to VL testing guidelines was hindered by ambiguities in VL ordering tools and delayed return of test results. Strategies are needed to improve adherence to VL testing including focus on lower-level health centers and younger patients, intuitive VL monitoring tools and adequate sample collection supplies, and point of care testing to shorten results turnaround time.


In the multivariate analysis, the time taken to arrive at the ART clinic , higher HIV viral load , and the presence of HIV drug resistance mutation  were associated with NVP plasma concentration. A significant proportion of patients receiving the NVP-based regimen had supra- and sub-therapeutic plasma concentrations. Higher HIV viral load and the presence of HIV drug-resistant mutations are important factors associated with NVP plasma concentrations.This study aimed to determine the association between the plasma concentration of nevirapine (NVP) and clinical outcomes. In this cross-sectional study, sociodemographic and clinical data were collected from 233 HIV patients receiving NVP-based first-line antiretroviral therapy (ART) regimens in Nairobi, Kenya. The mean age was 41.2 (SD ± 11.9) years. Fifty-four (23.2%) patients had virological failure (>1000 copies/mL), whereas 23 (9.9%) were infected with drug-resistant HIV strains. Eleven patients had nucleoside reverse transcriptase inhibitor resistance mutations, including M184V and T215Y, whereas 22 had non-nucleoside reverse transcriptase inhibitor resistance mutations, including G190A, K103N, V106A, Y181C, A98G, and Y188L. The median NVP plasma concentration was 6180 ng/mL (IQR 4444–8843 ng/mL), with 38 (16.3%) patients having suboptimal NVP plasma levels of <3400 ng/mL. The majority 23 of the 38 (60.5%) patients with NVP C Kenya initiated the World Health Organization test and treatment guidelines in 2015, which significantly increased the utilization and access to ART treatment. By the end of 2021, approximately 74% of adults in Kenya requiring ART received ART, with approximately 68% achieving virologic suppression. In this study, first-line ART contained a backbone of two nucleoside reverse transcriptase inhibitors  and one non-nucleoside reverse transcriptase inhibitor, either efavirenz or nevirapine (NVP).Human immunodeficiency virus (HIV) treatment remains complex and many factors must be considered for successful clinical outcomes. Antiretroviral therapy (ART) has been instrumental in reducing the burden of HIV.–6 Data show wider interpersonal variations in NVP plasma levels among HIV patients with treatment failure, and a large proportion fails to achieve a therapeutic window.,8 Although several reports have linked plasma NVP concentrations with varied treatment outcomes, in many developing nations, Kenya, including therapeutic drug monitoring, has not yet been integrated into HIV management programs.,10Optimal drug exposure is critical for ART administration. Prolonged treatment with NVP or its suboptimal exposure has been associated with significant risks of virologic failure and the emergence of HIV drug-resistant strains. These factors are partly due to differences in host genetics and drug metabolism, thus supporting the need for therapeutic drug monitoring to boost the clinical management of HIV. Nevirapine is characterized by a prolonged half-life elimination and a low genetic barrier to resistance, hindering its long-term therapeutic efficacy. Therefore, this study was conducted to determine the relationship between patients’ clinical outcomes, including the presence of HIV drug-resistant mutations, and NVP plasma concentration.Multiple factors contribute to ART treatment outcomes, including high interpatient variability.This cross-sectional study conducted between 2018 and 2019 enrolled 233 HIV patients receiving treatment at the Family AIDS Care and Educational Services (FACES), Kenya Medical Research Institute (KEMRI), Kenya. These patients met the following recruitment criteria: consented to participate, aged ≥ 18 years, and received NVP-based first-line antiretroviral (ARV) treatment for the past 12 months. The first-line ART at the time of this study comprised zidovudine, abacavir, TDF, stavudine, 3TC, and efavirenz or NVP (NVP). The ART regimen formulation and dosing used in this study were per the guidelines of the Ministry of Health, National AIDS, and sexually transmitted infection Control Program. based on population proportion estimation with specified relative precision. The alpha (α) was set at 0.05, relative precision (ε) at 0.20, and the prevalence of individuals on first-line ART was assumed to have drug-related toxicities as a result of abnormally high plasma concentrations of 18%. In total, 233 patients were recruited to achieve 0.95 power.The sample size was calculated using the formula described by Lemashow et al,Patient information related to this study was obtained through detailed face-to-face interviews. Approximately 8 mL of blood samples were collected 5 to 8 hours post ARV uptake into three blood tubes as follows: ethylenediaminetetraacetic acid anticoagulant tubes for immunological testing. Serum separation tubes for clinical chemistry, lithium heparin tubes for HIV viral load, and NVP plasma level quantification. The samples were stored at −80°C after collection and analysis. The biological samples were processed at the KEMRI-University of Washington Nairobi laboratory. A sample (whole blood and plasma) aliquot was sent to the Chemistry Laboratory of Jomo Kenyatta University for NVP plasma level validation. Adherence was measured based on dose compliance during the 30 days preceding the latest refill. The quantity of dose pills at refill was counted and reconciled against the dose counts dispensed at last refill. Additionally, pill count data were obtained from patient cards for the four months preceding the study period. Nonadherence was determined as the percentage of overdue dose at refill, averaged over a four-month period and used to assign adherence as good (≤5% dose skipped) while as poor (>15% dose skipped).Adherence to ARVs was measured by reviewing the patient medical cards, pharmacy refill data and medical records as described in our previous study Ngayo et al.Nevirapine (purity: 99.0%) and the internal standard, nevirapine D4 (purity: 99.3%), were purchased from Alsachim . A 200 μg/mL NVP stock solution was diluted with 50% methanol in water to a concentration range of 752.27 and 121,333.33 ng/mL. The internal standard was diluted with 50% methanol to obtain a working solution of 100 ng/mL. Then, 20 µL of the working standard and 20 µL of IS were further diluted in 200 µL of drug-free human plasma to prepare six plasma calibrators at a 10-fold dilution.,16 Briefly, a mixture of each plasma sample (50 µL) and internal standard (5 µL) in a 1.5 mL Eppendorf tube was heated for 10 minutes at 65°C and then cooled at room temperature for 10 minutes. One hundred microliters (100 µL) of cold methanol (−20°C) was then added and held cold for 10 minutes and subsequently centrifuged at 20,000 g at 20°C for 8 minutes, and the supernatant was collected in a clean 1.5 ml Eppendorf tube. Ammonium acetate buffer  was added to the supernatant and briefly centrifuged. The samples were inactive and safe for further processing in the inorganic laboratory.The method for viral inactivation has been described in our previous publications. modified in our previous publication.,16 Plasma samples were extracted using Bond Elut C18 cartridges, according to the manufacturer’s instructions . The eluents were completely evaporated using Thermo Scientific Reacti-Vap evaporators  at 37°C for 30 minutes. This was then reconstituted using 100 µL of equal parts 1:1 acetonitrile and water, vortexed briefly, transferred into 50 mL capped vials, and placed into Xevo TQ-S (Waters Corporation) for quantification. Approximately 1 µL of the sample was injected automatically into the LC/MS/MS instrument and quantified within 5 minutes. NVP plasma concentrations were categorized as <3400 ng/mL (below the therapeutic range), 3400 to 6000 ng/mL (therapeutic range), or >6000 ng/mL (above the therapeutic range).,19The NVP plasma concentrations were quantified using a tandem quadrupole mass spectrometer (LC/MS/MS) designed for ultra-high performance: Xevo TQ-S  as described by Reddy et al® . Assays were performed according to the manufacturer’s instructions.Cluster of differentiation 4 (CD4) cell counts were measured using a FACSCount TM flow cytometer , whereas HIV-1 RNA was measured using a Generic HIV Viral Load Resistance mutations were identified using Stanford University and International AIDS Society-USA website interpretation algorithms.The presence of HIV drug-resistant mutations was tested using an in-house genotypic method as previously described by Lehman et al.All the data were subjected to descriptive data analyses. Frequencies and percentages were used to present sociodemographic data. Steady-state NVP plasma concentrations were not normally distributed according to the Shapiro–Wilk test; hence, the Kruskal–Wallis test, Dunn’s test, and quantile regression analysis were used to evaluate the association with NVP plasma concentrations at the 5% significance level. All statistical analyses were performed using STATA v. 13 .2, with an average time taken to the ARV clinic of 83.4 (SD ± 47.1) minutes, and an average transportation cost to ARV clinic of 231.1 (SD ± 176.5) Ksh. The average duration of living with HIV was 6.4 (SD ± 5.7) years, with a majority of 160 (68.7%) of the participants taking ARV between 1 and 5 years, while 165 (70.8%) had changed their previous ARV regimen. There were 149 (63.9%) of the participant who had poor ART adherence rate.A total of 233 adult participants receiving a first-line ART regimen were evaluated, with a mean age (SD) of 41.1 (11.9) years. There were 144 (61.8%) females, 85 (36.5%) with a tertiary level of education, 148 (63.5%) were Bantus, 122 (52.4%) were unemployed, 193 (82.8%) were married, and 135 (57.9%) were currently living with their children. There were 105 (45.1%) participants who consumed alcohol, and only seven (3%) abused other drugs. The mean (SD) age of sexual debut was 17.6 (2.3) years, and 167 (71.7%) had the age of sexual debut of < 18 years. The mean (SD) for body mass index was 25.9 (4.9) kg/mThe mean (SD) CD4 count for the participants was 418.7 (357.5) cells/μL, with 162 (69.5%) having CD4 < 500 cells/μL. The mean (SD) viral load count for the participants was 17345.1 (89165.7) cells/mL with 179 (76.8%) of the participants being virally suppressed (<1000 cells/mL). Of the 233 participants enrolled, 23 (9.9%) harbored drug-resistant virus at the time of sampling, 26 (11.2%) and 52 (22.3%) had alanine aminotransferase and aspartate aminotransferase levels, respectively, and 67 (28.8%) had anemia  patients with NVP Cmin between 3400 and 6000 ng/mL or 2 of the 126 (1.3%) patients with NVP Cmin > 6000 ng/mL (P = .001).Approximately 23 patients (9.9%) were infected with drug-resistant HIV. G190ASEQ (3.9%) was the most common non-nucleoside reverse transcriptase inhibitor mutation, followed by K103NS 7 (3.1%), V106AM (1.3%), Y181 CIV, and V108IV, each occurring in 2 (0.9%) participants. The most common NRTI mutation was M184V, which occurred in 10 (4.3%) patients. Of the two patients who harbored TAMs mutations, all had T215Y mutations among those who were on 3TC/NVP/TDF treatment. The majority 23 of the 38 (60.5%) patients with NVP CP = .0001). The majority 126 (54.1%) of the participants had NVP plasma levels of > 6000 ng/mL, which are considered levels for durable viral suppression. There were 69 (29.6%) participants with NVP levels of < 3400 ng/mL considered levels for poor viral suppression and the least 38 (16.3%) had NVP levels between 3400 and 6000 ng/mL considered levels for viral mutant selection windows.The mean NVP plasma (SD±) titer was 7165.1 ± 4544.3 ng/mL ranging between 450 to 44207 ng/mL. The Shapiro–Wilk W test showed that the distribution of the NVP plasma levels was regular among the study participants . Participants with suboptimal NVP levels of < 3400 ng/mL had lower CD4 count 1 to 500 cell/µL compared to participants with NVP levels between 3400 and 6000 ng/mL or <6000 ng/mL (P = .022). Participants with suboptimal NVP levels of <3400 ng/mL were infected with virus with various resistant mutations than participants with NVP levels between 3400 and 6000 ng/mL or <6000 ng/mL (P = .0001). Participants with suboptimal NVP plasma levels of <3400 ng/mL had significantly higher viral load >1001 cells/mL than participants with NVP levels between 3400 and 6000 ng/mL or <6000 ng/mL (P = .0001). A trend was observed regarding participants with poor ART adherence with a higher percentage of them having lower NVP plasma level <3400 ng/mL when compared to participants with NVP levels = 3400 to 6000 ng/mL and NVP levels > 6000 ng/mL . There was no significant statistical difference observed regarding occurrence of adverse outcome such as skin rash, hemoglobin count, aspartate aminotransferase count, and alanine aminotransferase levels in participants with NVP plasma levels > 6000 ng/mL when compared to participants with lower NVP plasma levels  as compared to participants with NVP levels between 3400 and 6000 ng/mL or <3400 ng/mL (mL P = .02. ParticP = .031), higher HIV viral load , and the presence of HIV drug resistance mutation   were aged ≥ 31 years, implying that ART coverage among the youth population might still be a challenge in Kenya. The NAAC reports that initiating and continuing treatment is particularly problematic in adolescents and young people. In 2019, only 25% of HIV-infected adolescents received ART, of whom 66% were virally suppressed.In this study, men living with HIV were significantly less likely to be on treatment than women, which reflected the current ART treatment trend in Kenya, with 80% of women and 65% of men accessing ART treatment. reported fewer (6.2%) patients with sub-therapeutic NVP levels. Oluka et al reported even fewer patients had suboptimal NVP levels. Giacomelli et al, reported that 5.6% of patients had subtherapeutic NVP plasma concentrations. In contrast, Gopalan et al, reported a sub-therapeutic trough plasma NVP concentration in 65% of the children sampled in India.This study reported NVP plasma concentrations ranging from 450 to 44207 ng/mL. There were 38 (16.3%) participants with NVP levels <3400 ng/mL who were considered to have poor viral suppression. Other studies have reported varied outcomes, with Shapaya The study further showed that the frequency of quasispecies with K103N decreased over time, whereas the proportion of Y181C and G190A increased from 0% to nearly 100%. In the current study, the majority of patients with various drug-resistant mutations had NVP levels < 3400 ng/mL, indicating poor viral suppression. This is consistent with a study by Vardhanabhuti et al, which showed that women of African ancestry with HIV-resistance mutations had a lower median time to NVP IC50 than Indian women. In contrast, Oluka et al, reported no association between HIV drug resistance and plasma NVP levels in Kenyan women. Cumulatively, these results indicate a dynamic relationship between plasma NVP levels and the occurrence of drug-resistance mutations. Nonetheless, maintaining an optimal plasma concentration of NVP is critical because a single point mutation at a specific position on the pol gene in the HIV-1 genome may confer high-level resistance to NVP. Studies have indicated that a trough plasma NVP concentration (Ctrough) of/ = 3 μg/mL is associated with an increased risk of treatment failure by promoting the evolution of drug-resistant viral variants.The prevalence and types of drug-resistant HIV strain infecting patients in this study were consistent with previous reports that showed that during long-term ART treatment, the frequency of specific HIV drug-resistant mutations conferring resistance to NVP  were dynamic. showed that patients with virologic success had higher NVP plasma concentrations than those with virologic failure . The INCAS trial showed that NVP plasma concentration ranging between 3.45 and 3.88 µg/mL at week 12 was predictive of virologic success after 52 weeks of therapy. In our study, patients with virologic failure  had lower NVP plasma levels (<3400 ng/mL) than patients with NVP plasma concentrations > 3400 ng/mL. Our results are consistent with previous results obtained from clinical trials on Caucasians, Africans, and Chinese; higher NVP trough concentrations were associated with a lower rate of virologic failure. In a Chinese study, the median Ctrough levels among Chinese patients with partial and complete responses after 6 months of treatment were significantly higher than those among patients with viral failure. In contrast, a previous study in Kenya by Oluka et al, showed no association between plasma NVP levels and viral suppression.The relationship between the NVP concentration and virologic responses has been explored in various studies. The study by Duong et al, Studies have consistently shown strong associations between poor ART adherence and adverse clinical outcomes, including patient mortality and disease progression, as measured by CD4 cell count, viral load, and the development of drug resistance. Despite mounting evidence on the benefits of ART adherence, suboptimal adherence remains common in adults and children. This association has been attributed to an unstable steady state in the NVP levels due to poor adherence. This can be further explained by the negative correlation coefficient, whereas the number of days missing to take a dose increased, the plasma drug levels decreased. Different adherence measurement methods  have yielded varied results regarding treatment outcomes. In this study, we used patient medical cards, pharmacy refill data and medical records adherence measurement method that can face accuracy challenges especially if the ARV pills are not swallowed or taken in the presence of healthcare giver.Although adherence was not associated with NVP plasma concentrations in this study, ART adherence is generally recognized as an essential component of the successful management of HIV/AIDS. education level , and ARV adherence with the level of education. Smoking has been associated with enzyme induction, causing higher metabolism, and hence, reduced plasma NVP levels. In contrast, alcohol uptake is associated with higher plasma NVP levels, which is attributed to the reduced metabolism of NVP due to reduced metabolic enzyme concentrations due to reduced liver function. The previous uptake of the efavirenz-based regimen is associated with higher NVP levels given that the two drugs are metabolized by the same enzymes; hence, the reason for changing from efavirenz might have been due to reduced metabolism, which also affects NVP. The CD4 nadir level has also been shown to influence the NVP plasma level. Other factors include the presence of skin rash, co-treatment with other infections such as tuberculosis, meningitis, malaria, social challenges, and HIV-related disclosures.Other studies measuring factors associated with NVP plasma levels have identified other independent factors that we did not either measure or find to be significant in this study, including duration of therapy,This study reported highly heterogeneous NVP plasma concentrations, with a significant proportion of patients receiving the NVP-based regimen had supra- and sub-therapeutic plasma concentrations. Higher HIV viral load and the presence of HIV drug-resistant mutations are important factors associated with NVP plasma concentrations.The authors would like to thank the study patients enrolled in the FACES-KEMRI care and treatment program as well as the technical staff at KEMRI. We wish to acknowledge Director General KEMRI for allowing us to publish this work.Conceptualization: Evans Okumu Omondi, Anne Muigai, Musa Otieno Ngayo.Formal analysis: Juster Mungiria, Raphael Lihana.Funding acquisition: Musa Otieno Ngayo.Methodology: Evans Okumu Omondi, Anne Muigai.Supervision: Anne Muigai, Raphael Lihana.Validation: Evans Okumu Omondi, Musa Otieno Ngayo, Juster Mungiria.Writing – original draft: Musa Otieno Ngayo.Writing – review & editing: Evans Okumu Omondi, Anne Muigai, Juster Mungiria, Raphael Lihana.


Adherence to antiretroviral treatment (ART) remains the cornerstone of optimal HIV outcomes, including viral suppression (VS), immune recovery, and decreased transmission risk. For many people with HIV (PWH), particularly those with early-acquired HIV, structural, behavioral, and cognitive barriers to adherence and competing priorities related to life events may be difficult to overcome, resulting in nonadherence. Long-acting injectable antiretroviral therapies (LAI-ART) may be a useful strategy to overcome some of these barriers. However, to date, the approved LAI-ART strategies ) have targeted those who have already attained viral suppression, precluding their use in the 40% of adolescents and young adults (AYA) that VS has eluded.3 for > 5 years. She received a 1-month oral lead-in (OLI) of cabotegravir/rilpivirine, followed by the injectable loading dose. She has since adhered to all her monthly dosing appointments, sustained VS, and transitioned to a bi-monthly injection schedule.Ms. X is a 30-year-old woman with perinatally-acquired HIV and barriers to adherence. Despite many interventions, she remained persistently viremic, with resultant immune suppression and multiple comorbid opportunistic conditions, and viral load (VL) > 10,000,000 copies/ml. Given her longstanding history of poor adherence to an oral regimen, a switch to monthly intramuscular (IM) injections and biweekly infusions of ibalizumab were initiated leading to decreased viral load to 8,110 copies/ml within two weeks. Ms. H is a 33-year-old woman with cognitive limitations due to childhood lead poisoning. Her viral load trajectory took a downward turn, precipitated by various life events, remaining elevated despite intensive case management. Initiation of LAI-ART (CAB/RPV) in this patient led to an undetectable VL (< 20 copies/ml) within two months of treatment initiation. Miss Y. is a 37-year-old woman with perinatally-acquired HIV and chronic challenges with nonadherence and longstanding immunosuppression with CD4 < 200 cells/mmThese three individuals with HIV  with longstanding nonadherence and persistent viremia were successfully initiated on LAI-ART through the process of care coordination and the collective efforts of the care team, highlighting the barriers, challenges, and the multidisciplinary coordination needed to assure successful implementation of this strategy for the most vulnerable of patients. Antiretroviral treatment (ART) has transformed HIV infection from an acute life-threatening condition to a chronic manageable one and has reduced HIV/AIDS-associated deaths by nearly 70% since the beginning of the epidemic , 2. ART The recently approved long-acting injectable antiretroviral therapy (LAI-ART) formulations of cabotegravir, an integrase strand transfer inhibitor, and rilpivirine, a non-nucleoside reverse transcriptase inhibitor , have shLAI-ART may be a useful strategy to overcome the barriers posed by long-term oral ART. However, viral suppression remains a prerequisite for LAI-ART initiation to date, precluding its use in the 40% of adolescents and young adults (AYA) that VS has eluded . In thisThe patients described in this report are cared for in a youth-focused multidisciplinary care program, the Accessing Care Early (ACE) Clinic . EmbeddeMs. X is a 30-year-old African-American female with perinatally-acquired HIV. Her parents were HIV positive, and her mother passed away from complications of AIDS when she was 15 years old. Clinical records from when the patient was 11.8 years old demonstrate persistent fluctuations in her viral load with only brief episodic VS, last at 16.2 years. Combination therapies received in chronological order from the time of ART initiation were (1) stavudine (d4T), efavirenz (EFV), lopinavir/ritonavir (LPV/RTV); (2) atazanavir (ATV), ritonavir (RTV), emtricitabine/tenofovir disoproxil fumarate (FTC/TDF); (3) darunavir (DRV), RTV, FTC/TDF, raltegravir (RAL); (4) ATV, RTV, FTC/TDF, RAL; (5) ATV, RTV, FTC/TDF; (6) ATV, RTV dolutegravir (DTG), FTC/TDF; (7) DTG, FTC/TDF; (8) DTG, FTC/tenofovir alafenamide (TAF); and (9) DTG/RPV, DRV/cobicistat (DRV/c). During the course of treatment, ART regimens were modified based on virologic failure, resistance-associated mutations (RAMs), and for simplification. Her cumulative resistance included: nucleoside reverse transcriptase inhibitor (NRTI) RAMs L210W, T215E/D, K101E, Y181Y/C, and protease inhibitor (PI) RAMs L63P, A71T/A. No INSTI mutations were identified.Barriers to adherence included cognitive/developmental delay, lack of social support, housing instability, transportation difficulties, poverty, intermittent phone service, lack of childcare, pill burden, medication side effects, and internalized stigma. Various interventions were tried, including simplifying her ART therapy to a once-daily regimen, setting alarm clocks and pillboxes, arranging transportation, assisting with housing, providing adherence counseling, and directly observed therapy (DOT), and connecting her with a patient navigator to help with her outpatient care. Despite these interventions, she remained persistently viremic, even during her two pregnancies, with resultant immune suppression and multiple comorbid opportunistic conditions, including intestinal mycobacterium avium intracellulare (MAI), diffuse molluscum contagiosum, recurrent vaginal infections, and recurrent pneumonia prompting frequent hospitalizations. At 30.6 years of age, she was hospitalized for refractory hypotension in the setting of profuse diarrhea. Her viral load at this time was > 10,000,000 copies/ml. She was re-initiated on her home regimen of DTG/RPV + DRV/c, improving her viral load to 49,800 copies/mL within ten days. Given her longstanding history of poor adherence to an oral regimen, the severe consequences of her advanced immunosuppression, and her proven inability to adhere longitudinally to an oral regimen, the decision was made to switch to monthly intramuscular (IM) injections of CAB/RPV (in clinic), without the OLI, along with biweekly infusions of ibalizumab. Her ibalizumab was initially administered in the clinic and thereafter via home infusion. Her daily oral regimen of DRV/c was continued for additional coverage. Her viral load decreased from 49,800 to 8,110 copies/ml within two weeks. Her most recent viral load was 512 copies/ml four months after initiation of the hybrid regimen , her lowest viral load since the age of 16 years  CAB/RPV through the medical benefit and the state-funded copay assistance program, allowing the medication to be initiated promptly and without financial barriers.3), and growing concerns that the patient’s continued intermittent adherence to her oral ART would lead to INSTI resistance and preclude her from the use of currently approved LAI-ART, the decision was made to initiate LAI-ART (CAB/RPV) without OLI plus liquid DRV and RTV. Her viral load became undetectable (< 20 copies/ml) within two months of treatment initiation and has remained undetectable since then , and zidovudine (AZT) during pregnancy in 2011 but had to undergo a Cesarean section secondary to rising HIV viral load despite reported medication adherence. She was enrolled in the PROMISE study of ART in pregnancy and assigned to the no-treatment post-partum arm, leading to the temporary discontinuation of ART after delivery. She resumed ART a year later. All combination therapies received in chronological order from the time of ART initiation were (1) LPV/RTV, 3TC, AZT; (2) DRV, RTV, DTG, TDF/FTC; and (3) bictegravir (BIC)/TAF/FTC. During the course of treatment, changes were made to ART regimens based on virologic failure, resistance panels, and for simplification. Her cumulative resistance included: NRTI RAMs M184V and INSTI accessory resistance mutation G163R. Her viral load trajectory took a downward turn, precipitated by various life events, and exacerbated by depression, anxiety, panic attacks, and nausea and vomiting with medications unrelieved by prescribed antiemetics. Her viral load remained elevated despite intensive case management, including DOT and medication reminders. With her persistent viremia (8930 copies/ml), immune decline  for check-ins and assessment of adherence with adherence counseling. She too had Medicaid, which provided coverage for LAI CAB/RPV through the medical benefit and the state-funded copay assistance program. The patient was expeditiously started on the regimen as soon as the clinic had an available appointment.Miss Y. is a 37-year-old woman with perinatally-acquired HIV, initially diagnosed at five years of age when her mother died of HIV. She has had chronic challenges with nonadherence, which she attributes to limited support systems during her youth as well as currently with competing responsibilities , medication side effects , and internalized stigma and pill aversion when having to take her medication. Her HIV regimen history included (1) monotherapy zidovudine (AZT), (2) nelfinavir, stavudine, and lamivudine , (3) AZT, abacavir, lamivudine , (4) atazanavir, ritonavir, tenofovir, emtricitabine , (5) efavirenz tenofovir, emtricitabine , (6) darunavir, ritonavir, tenofovir, emtricitabine , and ultimately bictegravir, tenofovir, emtricitabine (BIC/TAF/FTC), all of which she took variably, with persistent intermittent nonadherence. During the course of treatment, ART regimens were modified based on virologic failure, resistance panels, and for simplification. Her cumulative resistance included: NRTI RAMs D67N, T69D, and NNRTI RAMs A98G. No INSTI mutations were identified. Given her nonadherence, she had longstanding immunosuppression with CD4 < 200 cells/mmImmune reconstitution inflammatory syndrome (IRIS) was not observed in any of the patients.This paper reports on three young PWH with poor adherence to oral ART and initial viremia being successfully managed with LAI-ART. Although many clinical trials have reported findings on the efficacy, tolerability, or acceptability of LAI-ART, this is one of the first case series that reports on the outcomes of LAI-ART use among patients facing adherence barriers and unable to viroologically suppress, in a clinical, non-research setting. The three patients presented here have unique circumstances and may not be represented by the experiences reported in the clinical trials. Specifically, the randomized clinical trial of cabotegravir and rilpivirine in treatment-experienced patients (ATLAS study) was limited to PWH who were adherent to ART, were virologically suppressed (VL < 50 copies/ml), and had not experienced virologic failure on oral regimens at least six months prior to study enrollment . These pThe need for LAI-ART for patients with adherence barriers to oral regimens is also demonstrated by the results of a recently published compassionate use initiative that allowed providers to request LAI-ART, cabotegravir, and rilpivirine (with or without the month lead-in), for their patients with adherence challenges mostly from psychologic conditions . NotablyOur outreach team contacted patients, ensured closed follow-up through calls and text messaging, provided transportation to get patients with financial or logistical constraints to their appointments promptly, and also facilitated family coordination. Collaboration between providers, laboratory staff, and home care fostered effective viral load monitoring, and funding support through the Ryan White program made the care coordination feasible. Lastly, medical insurance that covered the LAI-ART without significant barriers was key to accessing/securing the medications. The aforementioned collective effort and team coordination resulted in the successful treatment and improved clinical and virologic outcomes for these patients, demonstrating that PWH confronting adherence challenges can and do succeed on LAI-ART when purposive support is provided and strategies and systems of support are in place.


HIV viral load (VL) is an important indicator to monitor treatment response in antiretroviral therapy (ART). Patients on ART may experience viral blips, with low-level elevations of VL between 50 and 999 copies/mL known as low-level viraemia (LLV), but not reaching the threshold for virological failure  defined by WHO guidelines. The objective was to investigate the long-term impact of LLV on virological failure.We analyzed adults who were ART naïve at baseline. LLV was defined as having an VL of 51–999 copies/mL at least once. The subjects with LLV were grouped into three categories: 51–199, 200–399, and 400–999 copies/mL. Patients with multiple episodes of LLV were classified based on the highest VL result. The subjects with LLV were also grouped by the frequency of LLV, i.e., a single episode, two consecutive episodes, two intermittent episodes, more than two consecutive episodes, and more than two intermittent episodes. Multivariable Cox models were used to predict the association of LLV with virological failure.A total of 93,944 subjects were included. The median number of VL tests performed was 3. There were 21,203 LLV cases, with an overall incidence of 22.6%. Most of the LLV cases were found in subjects with LVs of 50–199 copies/mL, followed by 400–999 and 200–399 copies/mL. Most of the LLV cases experienced single episodes, and the numbers of LLV with two consecutive episodes, two intermittent episodes, more than two consecutive episodes and more than two intermittent episodes were decreased successively. The risk factors associated with virological failure include: intermediate-level (200–399 copies/mL) and high-level (400–999 copies/mL) LLV, single episodes of LLV and two or more than two consecutive episodes of LLV, which may put the subjects at a 1.28–2.26-fold higher risk for virological failure.Strengthened immediate medical attention should be placed on patients with VL of 200–999 copies/mL. The patients having experienced LLV once should be targeted for case management and repeat VL testing within 24 weeks to determine persistent LLV and monitor virological failure. The invention of antiretroviral therapy (ART) in 1996 led to significantly improved quality of life for persons infected with the human immunodeficiency virus (HIV) in the long term. Although ART cannot completely cure AIDS, it helps to suppress viral load (VL), maintain functions of the immune system and prevent disease progression to AIDS or death , 3. One Although optimal viral load suppression below LLOD is expected, some patients may experience virological failure (VF) or persistent Low-level Viraemia (LLV), i.e., those that fall between LLOD and VF. Although the ART clinical guidelines across the regions show clearly what targeted clinical interventions can be provided immediately to the patients who have been identified as VF, they have limited recommendations for clinical management of LLV cases. In addition, they define thresholds for virological failure differently. For example, the World Health Organization (WHO) definition of VF requires a VL of ≥1,000 copies/mL, but it also flags that VLs below 1,000 copies/mL typically suggest the risks for subsequent VF . China'sThe existing LLV studies were mainly conducted in developed countries in regions of Europe and North America –13 ratheYunnan, which lies in the southwest corner of China sharing border with the Golden Triangle in Southeast Asia, was a province hit hard by the HIV-1 epidemic with China's first HIV-1 cases diagnosed there . It has In this study, we conducted a multicenter, retrospective observational analysis of a large dynamic cohort of individuals enrolled between 15th April 2004 and 31st December 2018 and receiving ART at 235 HIV treatment centers in Yunnan, China. These HIV treatment centers include health care facilities at the four levels—provincial, prefecture/municipal, county/district, and township, as well as the ones in the confinement settings.All HIV treatment centers treated and monitored patients following China's National Free HIV Antiretroviral Drug Treatment Manual (Version 1–4). The first-line regimens consist of 2 nucleoside reverse-transcriptase inhibitors (NRTIs) and one non-nucleoside reverse-transcriptase inhibitor (NNRTI), and the second-line regimens include two NRTIs and a protease inhibitor (PI). See A total of 110,413 subjects ever enrolled in HIV treatment between April 15, 2004 and December 31, 2018 were screened, with 24,345 cases ruled out according to the following exclusion criteria: (1) 224 subjects who were initiated on treatment at an age of <15 or with unknown dates of birth; (2) 22,002 subjects who did not have any VL measurement; (3) 1,340 subjects who did not have any VL measurement 20 weeks after treatment initiation of the respective ART regimens; (4) 779 subjects whose initial therapy was either unknown or not in compliance with the standards, such as monotherapy, two-drug therapy, three-drug therapy with wrong combination, or any ART regimens not covered by the National Manual for Free HIV Antiviral Treatment for which patients had to pay out of pocket. In total, 86,068 subjects were included in the analysis.Recommended by China's National Free HIV Antiretroviral Drug Treatment Manual, the PI Lopinavir/Ritonavir (LPV/r) has been consistently used over the past years to replace the NNRTI for the switch to the second-line ART regimen see . For thiHistory cards of HIV patients as of December 31, 2018 were retrieved from the HIV/AIDS Comprehensive Response Information Management System (CRIMS) of the Chinese Center for Disease Control and Prevention. CRIMS database is real-world clinical data uploaded by all HIV treatment centers within 5 days following patient visits and is audited for data quality once a year.According to the national policy for free ART, each HIV patient can receive one VL test for free each calendar year, which means that a person with HIV will be tested free for VL at a visit ≥ 20 weeks after ART initiation and repeat the VL test once for each subsequent year. In reality, in addition to the single VL test provided free by the national program, additional VL tests may be ordered at the discretion of HIV care providers at each site with the testing cost covered by patients out of pocket or other funding resources. In case the time interval between two consecutive VL tests performed was <48 weeks, some patients may have more than one documented VL result for every 48 weeks of staying on ART. In this study, VL testing was to detect the numbers of HIV-1 RNA nucleic acid in peripheral blood plasma. VL testing was performed in the laboratories of designated HIV treatment centers at the provincial and prefecture levels. Blood samples collected by the county and township-level HIV treatment centers were separated, sub-packaged, frozen and transported to the laboratories of the prefecture-level HIV treatment centers for VL testing.Over time, the sensitivities of the VL assays in Yunnan have been improved. Since the inception of the cohort in 2004, the HIV laboratories in the province mainly used the COBAS AmpliPrep & COBAS TaqMan 48/96 (Roche Molecular Diagnostics), the VERSANT 340/440 Molecular System bDNA (SIEMENS), and the NucliSens EasyQ Analyzer (bioMérieux) for VL assays assay. Prior to 2011, most assays used in the province had a threshold of detection that is <400 copies/mL, and a small number of assays for research or epidemiological surveillance had a higher VL sensitivity of <50 copies/mL. After that, VL assays in Yunnan were gradually upgraded with a lower detection limit of <50 copies/mL or <20 copies/mL. For All VLs, the results that were below the LLOD or had “Target Not Detected (TND)” displayed were recorded as 0 and all the quantifiable VL measurements were recorded as numerical values.In this study, the start points of the two ART cohorts for observation were the dates of treatment initiation, i.e., the specific dates when the study participants were initiated on the first-line ART for the first-line ART group and the dates when LPV/r was first used in treatment for the second-line ART group. The primary endpoints for the two ART groups were when the study participants experienced VF. This study defined VF as having a first VL of ≥1,000 copies/mL at a visit ≥20 weeks after ART initiation or when switching to a LPV/r-based regimen. For the first-line ART group, the secondary endpoint refers to a patient switching to the protease inhibitor LPV/r-based regimen.The event for observation was occurrence of LLV. In this study, the subjects who had at least one HIV VL measurement between 50 and 999 copies/mL at visits ≥20 weeks after ART initiation were defined as LLV. DHHS, EACS, China's National Manual for Free HIV Antiviral Treatment, and WHO used different thresholds to define VF, i.e., >200 copies/mL, 400 copies/mL and 1,000 copies/mL –10. For The incidence rate for LLV was defined as the number of the subjects who had at least one episode of LLV in the study participants provided with the first-line or second-line ART regimen over the total observation time, and then stratified by calendar or follow-up year. The incidence rate for VF was calculated as the number of patients with VF, among the study participants provided with the first-line or second-line ART regimen over the total observation time, and was stratified by calendar or follow-up year.t-test and Kruskai-Wallis rank sum test were used for normal and non-normal distributions, respectively after a normality test; and χ2 test was applied for categorical variables. Test level α = 0.05; for the calculation of the incidences of LLV and VF by follow-up year, the person-year incidence were reported with their 95% confidence intervals (CIs). In order to avoid bias by repeated testing in the same follow-up year, the first VL measurement of the year was used for analysis; and the multiple LLV episodes taking place in the same natural year were all included for the calculation of the natural-year incidence rate for LLV; After validation, the Cox proportional hazards model was used to assess the effect of LLVs on outcome events. The Cox analyses included the subjects who had been followed up for at least 48 weeks without experiencing VF during the same period and had two or more documented LV measurements, and excluded those who had VF for the initial VL measurement from analysis. At the time points of a subject experiencing VF or switch to second-line regimens with LPV/r, or the last VL measurement before the end of the observation point, right censoring was applied; Kaplan-Meier curves were used to show the effects of different values and frequencies of LLV occurrence on outcomes events.The history cards of the subjects were loaded into the R3.5.0 program. The unique ART codes for HIV patients that contain no personally identifiable information were used as the primary key for data analysis. Demographic characteristics and VL measurements of the subjects are presented using descriptive statistics, with standard deviations for normal distribution and medians and interquartile ranges (IQR) for non-normal distribution, respectively. In terms of group comparisons, for continuous variables, a Student's Among the 86,068 subjects included in the analysis, 65,688 received the first-line ART , 5,667 received second-line ART , and 14,713 received not only first-line but also second-line ART . Of the 14,713 subjects who received both types of ART, the individuals who met the inclusion criteria were put into the two respective ART groups for their corresponding stages of the HIV treatment, i.e., 11,048 subjects for the first-line ART group and 11,541 for the second-line ART group. In total, there were 76,736 subjects in the first-line group and 17,208 subjects in the second-line group .In the first-line ART group, male subjects outnumber females. In the second-line ART group, female subjects outnumber males. Both groups were dominated by individuals who were married or living with a partner. The majority of the subjects in the two groups were associated with heterosexual transmission. Compared with the first-line ART group, patients in the second-line ART group tended to have initiated on treatment at a younger age (37 years vs. 34 years) and with a higher CD4 count at treatment initiation (247 cells/mL vs. 253 cells/mL), and have experienced a higher CD4 increase after ART (166/mL vs. 204/mL). The median follow-up was 184 weeks (IQR 94–305 weeks) for the first-line ART group, and 156 weeks (IQR 78–256 weeks) for the second-line ART group .The median number of VL testing performed for each subject was 3 times for the two groups. The maximum number of VL tests was 23 for the first-line ART group and 16 for the second-line ART group .A total of 21,203 LLV cases were identified in the two ART groups. Specifically, 23.2%  of the subjects in the first-line group and 19.6%  of those in the second-line groups developed LLVs. Among the LLV cases, 26.4% for the first-line group  and 20.4% for the second-line group  experienced two or more episodes of LLV, respectively. The 76,736 subjects of the first-line ART group were followed for a total of 316917.9 person-years, with an LLV incidence of 6.63 per 100 person-years ; the 17,208 subjects of the second-line ART group were followed for a total of 56,446.64 person-years, with an LLV incidence of 5.97 per 100 person-years . On the numerical level, the majority of the LLV cases had an LLV that was between 51 and 199 copies/mL (59.1% in the first-line group vs. 60.3% in the second-line group), followed by the subjects with an LLV of 400–999 copies/mL (21.8% in the first-line group vs. 19.9% in the second-line group) and the ones having an LLV of 200–399 copies/mL (19.1% in the first-line group vs. 19.8% in the second-line group); in terms of frequency of LLV, most of the LLV cases were found having a single episode (73.6% in the first-line group vs. 79.6% in the second-line group), and the proportions of LLV subjects experiencing two consecutive episodes, two intermittent episodes, more than two consecutive episodes, and more than two intermittent episodes declined sequentially .n = 93,944) of the two ART groups experienced VF with at least one VL of ≥1,000 copies/mL. Specifically, 12,854 subjects (16.8%) in the first-line group experienced VF, and 7,196 (56%) of them were identified on their first VL measurement when they failed, and the remaining 5,658 (44%) on the subsequent VL measurement. In contrast, 3,304 subjects (19.2%) in the second-line group developed VF, with 2,085 (63.1%) and 1,219 (36.9%) subjects found on their first and subsequent VL measurement, respectively  of the subjects , whereas the incidence of VF among the subjects with LLVs of 51–999 copies/mL increased up to 2.49 per 100 person-years (95% CI 2.39–2.58). The subjects whose LLVs were between 51 and 199 copies/mL had an incidence of 1.77 per 100 person-years, which remained the same level as those who had never experienced LLV. For the subjects whose LLVs were between 200 and 399 copies/mL or between 400 and 999 copies/mL, the incidences of VF reached 2.79 per 100 person-years (95% CI. 2.56–3.03) and 4.12 per 100 person-years (95% CI 3.86–4.38), respectively.In the second-line ART group, the incidence of VF was 2.13/100 person-years (95% CI 1.99–2.27) for the subjects without LLVs, and increased up to 2.90/100 person-years (95% CI 2.63–3.19) for those whose LLVs were between 51 and 999 copies/mL. Specifically, the incidence of VF was 2.14/100 person-years (95% CI 1.85–2.47) among the subjects with LLVs of 51–199 copies/mL, which stayed the same level as the rate for the ones who had never developed LLV. In contrast, significantly increased incidences of VF were observed among the two groups whose LLVs were either between 200 and 399 copies/mL or between 400 and 999 copies/mL, i.e., 3.16 per 100 person-years (95% CI 2.54–3.89) and 5.04 per 100 person-years (95% CI 4.24–5.94), respectively .In the first-line ART group, the incidence of VF was 1.82/100 person-years (95% CI 1.76–1.88) among the subjects without LLVs. It increased gradually with the increased frequency of LLV occurrence—a single episode, two consecutive episodes, and more than two consecutive episodes, i.e., 2.61 per 100 person-years (95% CI 2.49–2.73), 2.80 per 100 person-years (95% CI 2.51–3.12), and 3.50 per 100 person-years (95% CI 2.94–4.13), respectively. For the subjects who experienced intermittent LLVs, that is, having an VL of <50 copies/mL between LLVs, the incidence of VF was similar to that in the cases without LLV, with no significant difference observed.In the second-line ART group, the incidence of VF was 2.13 per 100 person-years (95% CI 1.99–2.27) among the subjects who had never developed LLVs. The rate was increased significantly for the cases who had experienced a single episode of LLV, two consecutive episodes and more than two consecutive episodes, i.e., 3.08 per 100 person-years (95% CI 2.76–3.42), 2.96 per 100 person-years (95% CI 2.15–3.97) and 2.93 per 100 person-years (95% CI 1.42–5.33), respectively. However, the incidence of VF in cases with intermittent LLV, that is, having an VL of <50 copies/mL between LLV episodes, was similar to that among the subjects who never experienced LLV, with no significant difference observed .In the first-line ART group, 6,405 patients who experienced VF on the first VL test, 12,779 patients who had only one documented VL result, and 694 patients who had been followed up for <1 year were excluded. A total of 56,858 cases were included in the COX proportional hazards regression analysis of LLV and VF occurrence. In the second-line ART group, 1,998 who had VF on the first VL test, 2,637 with only one VL result, and 240 followed up for no more than 1 year were excluded, with a total of 12,333 subjects included in the COX regression analysis.The multivariate COX regression analysis for LLV level and VF suggests the following results: In the first-line ART group, the risk for VF among subjects whose LLVs were between 50 and 199 copies/mL was no different from that in the cases without LLVs. The subjects with LLVs of 200–399 copies/mL and 400–999 copies/mL had a greater hazard for VF, i.e., 39 and 102% higher than the ones without LLVs, respectively. Data for the second-line ART group suggested similar trends: there was no major difference in the risk of VF between the subjects with LLVs of 50–199 copies/mL and the ones who never developed LLV, and the risk of VF in subjects with LLVs of 200–399 copies/mL and 400–999 copies/mL was 33 and 126% higher than those who had no experience of LLV , Table 5p < 0.001), two consecutive episodes of LLV , and more than two consecutive episodes of LLV  had correspondingly higher risks of VF, respectively; In the second-line ART group, the subjects who developed LLV just once , or twice in a row  were at increased risk for VF, respectively, compared with the ones who never experienced LLV  and of VF were the highest over the first year of ART and gradually declined in the subsequent years. The yearly trend for VF in this study resonates with the findings from previous studies . HoweverIn this study, moderate and high levels of LLV, single episodes of LLV, two consecutive episodes of LLV and more than two consecutive episodes of LLV were associated with a 1.28–2.26-fold higher risk of VF, after controlling for covariates including sex, baseline CD4 count, marital status, HIV acquisition category, age, and the year of ART initiation. A large systematic review of observation of persistent LLV in persons living with HIV reported that persistent LLV can be an independent predictor of VF and that the presence of persistent LLV should not be explained solely by variation in assay characteristics and require further investigation . Most ofIncreased incidence of subsequent VF in subjects with LLV of 50–199 copies/mL was not observed, indicating no association between low-level LLV and VF. In contrast, the incidence of VF was increased in subjects with intermediate- (200–399 copies/mL) and high-level (400–999 copies/mL) LLV, resulting in an increasingly higher risk of subsequent VF. This conclusion echoed with the DHHS guidelines and the EACS guidelines for defining a VL of ≥200 copies/mL as the virologic threshold for VF. As evidenced by more studies and clinical practice, it is recommended that a lower threshold VL for VF should be considered in the WHO guidelines and China's National Manual for Free HIV Antiviral Treatment in order to endorse timely medical intervention for the patients with VLs of 200–1,000 copies/mL.Without waiting for the final revision of the definition of VF in the WHO guidelines and the National Free AIDS Antiretroviral Drug Treatment Manual, the HIV care providers in Yunnan and any other high-HIV-prevalence settings could give immediate medical attention to the HIV patients on ART who initially experience LLVs of 200–999 copies/mL, assess patient adherence and provide targeted education, counseling and support through HIV case managers . ConstraResearch design of this study was focused on grouping of the subjects by HIV RNA level and frequency of LLVs. Without cross cluster analysis of these two categorical variables, we could not generate findings in clearer ways to pinpoint targeted interventions for HIV patients; This observational study, which was based on real-world data, lacked randomization and could be biased in selection and exclusion of research participants.More than 20,000 individuals were excluded from data analysis due to the absence of documented VL testing results although there was statistical difference between the excluded subjects and the study participants in terms of their demographic characteristics. Compared with study participants, the subjects who were excluded from data analysis tended to be older in age (39.4 years vs. 37.2 years), had lower levels of CD4 count at baseline (237 copies/mL vs. 252 copies/mL), had a higher percentage of males (68.2% vs. 60.7%), and included more people with HIV enrolled in ART since 2016. Among the HIV patients initiated on ART since 2016, a large number of HIV patients were treated for <48 weeks, and had no documented VL results, constrained by the national HIV/AIDS treatment policy of one free VL testing each calendar year. The impact of missing values for the excluded subjects on the findings was unclear.Previous studies suggested that a VL of >99,999 copies/mL at baseline was associated with a higher risk for episodes of LLV . In thisThe retrospective observational analysis of the large dynamic cohort of HIV ART in Yunnan province identified frequent occurrence of LLV. In conclusion, our results suggest that single or consecutive episodes of intermediate- or high-level LLV predicts increased risk for VF, which necessitated further investigation and immediate interventions accordingly. This study forms the basis for development of evidence-based management of HIV patients with LLV in Yunnan.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study was approved by the Medical Ethics Committee, Yunnan Provincial Hospital of Infectious Disease (Approved Document No. K201913). In this study, the rights and interests of the participants were fully protected, with no potential risk posed to the subjects. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.JA, YL, and JL designed the study and developed the research protocol and tools. JA and JL were responsible for data collection, analyzed the data, interpreted the results, and drafted the manuscript with guidance from ST, TL, and XD. ST reviewed and contributed to editing the manuscript. All authors have read and approved the final manuscript.This project has been supported with the Joint Fund for Applied Basic Research (No. 2020202001AY070001-105) from the Yunnan Provincial Department of Science and Technology, Kunming Medical University, the Yunnan Infectious Disease (HIV/AIDS) Clinical Trial Research Center Project (202102AA310005-031), and China Free HIV Antiretrovial Treatment Program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Twenty-one months following the switch, all but one subject maintained their viral load < 50 cp/mL. One subject had two viral blips. For the entire population, the sCD163 values increased significantly from baseline  and from 6 months after the switch. The other markers did not change. After 6 months, the sCD163 increase was more pronounced in subjects with greater likelihood of immune activation  Conclusions: cART simplification to dual therapy was associated with macrophage activation despite successful virological control after almost two years’ follow-up. This was more pronounced in those at risk of immune activation.Objective: Immunadapt is a study evaluating the impact of combination antiretroviral treatment (cART) simplification on immune activation. We previously showed that switching to dual therapies could be associated six months later with macrophage activation. Followup continued up to 24 months after treatment simplification. Materials and Methods: Immunadapt is a prospective single arm study of successfully treated subjects simplifying cART from triple to dual regimens. Before cART change, at 6 months, and between 18 and 24 months following the switch, we measured IP-10, MCP-1, soluble CD14 (sCD14), soluble CD163 (sCD163), and lipopolysaccharide binding protein. Patients were stratified according to lower or greater likelihood of immune activation . Variables were compared using matched Wilcoxon tests. Results: From April 2019 to September 2021, 14 subjects were included (mean age 60 years, 12 men, 26 years since HIV infection, CD4 nadir 302 cells/mm The life expectancy of people living with HIV (PLH) has dramatically improved since the introduction of triple-drug antiretroviral regimens .However, the long-term potential side effects of combination antiretroviral therapy (cART) have led to the introduction of guidelines recommending dual therapies, generally consisting in the association of an integrase inhibitor with another molecule, such as a nucleoside- or a non-nucleoside transcriptase inhibitor. Dual therapies have proven their non-inferiority in terms of virological control when compared to triple c-ART ,3,4,5.However, data on the consequences on the inflammatory biomarkers of switching to dual therapies are contradictory, and few studies have focused on the effect on the immune activation of such a switch. The TANGO and SWORD trials, together with a recent systematic review, found no consistent pattern of change in inflammatory biomarkers with two-drug regimens ,7,8. HowImmunadapt is a prospective study aiming to assess the impact on immune activation and inflammatory markers of switching from a triple-drug to a dual-drug therapy. Followup was continued up to 2 years, and the results are shown here. Immunadapt is a single-arm prospective study initiated in April 2019. Its aim is to measure the impact on immune activation markers of switching from a triple-drug to a dual-drug regimen. Patients were selected among those routinely followed in the Department of Internal Medicine in Cannes General Hospital and the Department of Infectious Diseases in Nice University Hospital.Each patient meeting the inclusion criteria was offered to participate in this study.Recruitment continued until achieving 20 subjects, which was the sample size estimated necessary for the analysis. © viral load or Aptima HIV Quant Dx, Hologic, Tremblay-en-France, France), switching from a triple-drug to a dual-drug regimen as a simplification strategy, were included.HIV-1 infected subjects on stable and successful cART , and patients gave written informed consent to participate.The study was initially designed for a follow-up period of 6 months. However, based on our findings at that point, , it was Measurement of immune activation was performed just before the cART switch, approximately 6 months later, and between 18 and 24 months later. IP-10 and MCP-1 were measured using ProcartaPlex ImmunoAssays , while sCD163 and sCD14  and lipopolysaccharide binding protein  were measured with ELISA kits. As the study was initially designed for a 6-month follow-up, samples collected at inclusion and 6 months after the switch were frozen at −20° and were all analyzed simultaneously. An extension of the study was subsequently requested, and samples collected between 18 and 24 months were analyzed at a later date.Demographic and main viro-immunological characteristics were collected. Prior antiretroviral therapy was also recorded. Regimens were classified according to the following categories: nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), integrase strand transfer inhibitor (INSTI), and protease inhibitors (PI).During follow-up, plasma viral load was quantified 2 months after the switch. Viro-immunological parameters were measured, together with immune activation markers, approximately 6 months after treatment simplification and at the end of follow-up, i.e., between 18 and 24 months from the switch. Viral blips were defined as transient low-level viremia (LLV), between 50 and 500 copies/mL, preceded and followed by suppression, i.e., below the quantification limit of the assay . To asseDuring follow-up, clinical events and newly prescribed co-medications were also recorded. The main demographic and viro-immunological characteristics of the population are described. Baseline was defined as the day of the switch from a triple to a dual cART regimen. Differences in parameter values were measured from baseline to 6 months from the switch, from 6 months to 18–24 months, and from baseline to 18–24 months.We first measured changes from baseline to 6 months and 18–24 months after treatment simplification for the entire study population. Considering the small sample size and the fact that CD4/CD8 ratio at inclusion and IP-10, MCP-1, and LBP trajectories did not follow a normal distribution , variables were compared using the matched Wilcoxon’s test.3), prior AIDS-defining condition, or VLLV during follow-up [Patients were then stratified into those with lower or higher likelihood of immune activation, the latter defined by at least one of the following parameters: low CD4 nadir  and 14 of them continued to participate for a mean period of 21 months , requiring a switch of therapy to a triple-drug regimen. The six individuals not completing the extended follow-up period maintained their viral load below 50 cp/mL all along the 21 month-period after the switch (data not shown).p = 0.003) and from 6 months after the switch .In a population of successfully treated subjects switching from a triple-drug to a dual drug-HIV regimen as a simplification strategy, we found a significant increase in macrophage activation. Such immune activation continued to increase after a follow-up of almost 2 years and was higher in patients with a low CD4 nadir, a previous AIDS-defining event, or detectable residual viremia during follow-up. Macrophage activation has been linked to several non-AIDS-related complications, such as neurological, cardiovascular, and hepatic disorders ,16,17,18In particular, sCD163 is considered one of the best markers for atherosclerotic plaque formation in HIV-infected patients as well as among the general population . MoreoveInterestingly, the ANRS-ETRAL trial already showed an increase in sCD163 when switching from a boosted PI-containing regimen to Raltegravir and Etravirine dual therapy . In addiThe reasons for such macrophage activation in our study require clarification. As LBP values did not correlate with the trajectory of sCD163, we do not think that microbial translocation could be associated with such macrophage activation. We suggest at least two hypotheses: the first one is that the relatively low penetration of integrase inhibitors in lymph nodes, associated with the suspension of one NRTI, could be associated with insufficient drug pressure in reservoirs, thus facilitating the resurgence of HIV replication . The secAs has been reported from previous studies, including ours ,24, we fThe limitations of this study include the small number of subjects and its single arm design. A comparative arm with patients continuing a triple-drug regimen would shed more light on the effect of treatment simplification on immune activation. However, as no other medical interventions were recorded during the follow-up, in our view, it is unlikely that the trajectory of sCD163 could be explained by other factors than treatment modification. With the absence of a comparative arm evaluating the trajectory of sCD163 in subjects not changing therapy, we could say that its increase is associated with physiological aging. However, in our view, this hypothesis is very unlikely, considering the important increase we found over a short period, and also that Kroeze et al. did not find any change in sCD163 in patients on stable treatment . In addiIn conclusion, simplification to dual-drug HIV therapy was associated with macrophage activation despite successful virological control after almost 2 years’ follow-up. Such an increase was higher in subjects with greater likelihood of immune activation. Further studies should shed more light on the long-term clinical impact of such macrophage activation.


Access to antiretroviral therapy (ART) led to epidemiological changes in human immunodeficiency virus (HIV) associated lymphoma in high-income countries such as reductions in diffuse large B-cell lymphoma (DLBCL) and stable or increased Hodgkin lymphoma (HL) and Burkitt lymphoma (BL). In 2016, Malawi implemented a universal ART (UART) policy, expanding ART eligibility to all persons living with HIV (PLWH). We compare the distribution of lymphoma subtypes and baseline HIV and prognostic characteristics for lymphoma patients in Malawi before and after implementation of UART. We enrolled patients with pathologically confirmed incident lymphoproliferative disorders into a observational clinical cohort. At diagnosis, a comprehensive clinicopathological evaluation was performed. Of 412 participants, 156 (38%) were pre-UART (2013-June 2016) and 256 (62%) post-UART (July 2016–2020). HIV prevalence was 50% in both groups. The most common pre-UART diagnoses were DLBCL [75 (48%)], low-grade non-Hodgkin lymphoma (NHL) [19 (12%)], HL [17 (11%)] and, BL [13 (8%)]. For post-UART they were DLBCL [111 (43%)], NHL [28 (11%)], BL [27 11%)] and, HL [20 (8%)]. Among PLWH, 44 (57%) pre-UART initiated ART prior to lymphoma diagnosis compared to 99 (78%) post-UART (p = 0.02). HIV-ribonucleic acid was suppressed <1000 copies/mL in 56% (33/59) pre-UART and 71% (73/103) post-UART (p = 0.05). CD4 T-cell counts were similar for both groups. We observed similar findings in the subset of participants with DLBCL. Overall, there were no significant changes in incident lymphoma subtypes (p = 0.61) after implementation of UART, but HIV was better controlled. Emerging trends bear monitoring and may have implications for prognosis and health system priority setting.Trial registration: ClinicalTrials.gov identifier:NCT02835911. Human immunodeficiency virus (HIV) associated lymphoma has emerged as a leading cause of cancer-related mortality  for peopThe advent of antiretroviral therapy (ART) led to changes in the epidemiology of HIV-associated lymphoma in high-income countries , 3, 7–12The pathophysiology of HIV-associated lymphomas is heterogeneous. Although the etiological link between lymphoma and HIV is not fully understood, evidence shows that DLBCL occurs most commonly in individuals with advanced acquired immunodeficiency syndrome and severe immunosuppression , and is In sub-Saharan Africa, available data is mostly inconsistent with trends observed in high-income countries. ART access has increased significantly in the past 15 years . HoweverIn Malawi, HIV prevalence is 8.9% , making The KCH Lymphoma Study is a prospective, observational cohort of lymphoma patients receiving treatment according to local standards of care in Lilongwe, Malawi. KCH is the main referral center for cancer treatment for the central and northern regions of Malawi, serving approximately 10 million people. The study was initiated in June 2013 and is one of the largest prospective cohorts of lymphoproliferative disorders in the region. The study was approved by the University of North Carolina Institutional Review Board and Malawi National Health Sciences Research Committee. All patients provide written informed consent prior to enrolment. Patients aged five and over with newly diagnosed pathologically confirmed lymphoproliferative disorders, who intend to complete treatment at KCH are eligible to be enrolled. All cases are diagnosed with real-time evaluation through weekly telepathology conferences involving two to five pathologists in Malawi and the United States. The pathologists render a consensus diagnosis using local tissue biopsy and immunohistochemistry as previously described , 32. In At lymphoma diagnosis, a comprehensive baseline clinical and laboratory evaluation was performed for each participant, as described in detail previously . StagingThe funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.From 2013 to 2020, 412 participants were identified for the analytic cohort. One hundred and fifty-six (38%) participants were enrolled during the pre-UART period and 256 (62%) were enrolled during the post-UART period. The pathological distribution of lymphoma subtypes and HIV status is depicted in Baseline demographic and clinical characteristics of the cohort are depicted in PLWH in the pre-UART group had been on ART for a median of only 16.8 monthsat the time of enrollment  compared to those in the post-UART group who were on ART for a median of 31.1 months(p = 0.02). In the pre-UART period, fewer PLWH [44 (57%)] were initiated on ART before lymphoma diagnosis compared to PLWH in the post-UART group [99 (78%)] (p = 0.02). Median CD4 T-cell count for PLWH at lymphoma diagnosis was 142 cells/μL (IQR 82–284) pre-UART and 156 cells/μL (IQR 96–273) post-UART (p = 0.54). PLWH in the post-UART period presented with lower HIV ribonucleic acid (RNA) [0 log copies/ml (IQR 0–3.6)] than those in the pre-UART period [2.4 log copies/ml (IQR 0–4.2)] (p = 0.07). Fewer PLWH in the pre-UART group [33 (56%)] had suppressed HIV RNA <1000 copies/ml compared to those in the post-UART period [73 (71%)] (p = 0.05).Baseline clinical and pathological characteristics are depicted in A strong association between HIV and lymphoma has been noted since the beginning of the AIDS epidemic . While hThe most common lymphoma subtypes identified in our study cohort were DLBCL, low-grade NHL and BL, for both the pre-UART and post-UART periods, consistent with results from a study by Mezger et al.  for a suOur findings are also inconsistent with trends observed in the United States, where ART expansion was associated with declines in HIV-associated lymphoma , 41. ThiStudy strengths include high quality pathologically confirmed diagnoses using an innovative telepathology platform and recruitment of participants with prospective collection of data from the main cancer treatment facility in Malawi which serves approximately 60% of the country’s population. Our study also has some limitations. First and most critically, less than four years have elapsed from the start of the universal ART to the end of enrollment for this study. Therefore, it may be too early to reflect changes in lymphoma subtypes; more significant differences may be seen after more time has elapsed. Second, our study is based on a clinical cohort at a national teaching hospital. It is not reflective of a population-based cancer registry and therefore we cannot discern epidemiological trends that may exist at a national level. In addition, our cohort size is small compared to other studies that examine lymphoma epidemiology, limiting statistical power. Finally, our study lacks the ability to control for other cancer-related lifestyle risks such as tobacco use, diet, and alcohol consumption.In conclusion, we found no significant differences in incident lymphoma subtypes in our study cohort when comparing the pre-UART and post-UART periods in Malawi. However, HIV control was improved among PLWH and lymphoma in the post-UART period but without significant improvement in baseline CD4 T-cell counts. Lymphoma remains a public health challenge in sub-Saharan Africa. As ART scale-up continues, it will be important to continue monitoring emerging epidemiologic trends related to lymphoma, as well as to continue efforts to provide early and effective ART to PLWH to reduce lymphoma risk and decrease cancer-attributable morbidity and mortality.