Virological Non-Suppression among Newly Diagnosed HIV-Positive Individuals on Dolutegravir-Based Antiretroviral Treatment in Eastern Ethiopia: Follow-Up Study

There have been limited studies linking baseline factors, including the viral load (VL) test, with virological non-suppression since the introduction of dolutegravir (DTG)-based regimens as first-line antiretroviral treatment (ART) in Ethiopia. This study aimed to identify baseline factors associated with virological non-suppression between October 2020 and July 2022. A follow-up study was conducted in eastern Ethiopia among newly diagnosed people living with HIV (PLHIV). A questionnaire and a checklist were used to collect the data. Five milliliters of venous blood were obtained at baseline and six months to determine the VL. A VL test was performed using the Abbott RealTime HIV-1 assay. To determine predictors of virological non-suppression, bivariate and multivariate logistic regression analyses were used. There were 235 PLHIV enrolled, 70.6% of whom were female, with a mean age of 33.9 years. Of the 161 retained on ART, virological non-suppression was 8.7% at six months. Baseline predictors of virological non-suppression were age ≤ 30 years, a history of substance use, and a VL greater than 4-log10 copies/mL. In this cohort, virological non-suppression was found to be optimal but still lagged slightly behind the third 95%–target. Thus, targeted interventions, the introduction of baseline VL testing to improve treatment outcomes, and fostering the attainment of UNAIDS 95–95–95 targets are recommended. Furthermore, broader research is recommended to explore the reasons for virological non-suppression in the study area.


Introduction
The human immunodeficiency virus (HIV) is a significant threat to public health. Africa is the most severely affected continent, accounting for more than two-thirds of all PLHIV [1]. In Ethiopia, it was estimated that approximately 610,000 people were living with HIV in 2021 [2]. The Joint United Nations Programme on HIV/AIDS (UNAIDS) and the World Health Organization (WHO) encouraged nations and global partners to adopt a series of ambitious strategic target-setting initiatives aimed at eliminating the HIV/AIDS epidemic and improving patients' access to ART across the world [3]. One initiative will be to reach the UNAIDS 95-95-95 goal, which states that 95% of people with HIV should know their status, 95% of those diagnosed should receive continuous ART, and 95% of those on ART should have a suppressed viral load by 2030 [4]. In 2021, among people accessing treatment, 92% and 68% of all HIV-positive individuals worldwide had viral suppression [1]. In Ethiopia, by the end of the same year, 84%, 78%, and 75% of PLHIV were aware of their HIV status, were on ART, and had achieved viral suppression, respectively [2].
It is well known that virological non-suppression has become a common public health challenge in several African countries due to various multidimensional factors [5]. The

Study Setting and Period
This cohort follow-up study was conducted in 15 selected public health facilities (8 hospitals and 7 health centers) in eastern Ethiopia (Figure 1). The enrollment for the baseline study was conducted between October 2020 and July 2022. The criteria used to select health facilities were the presence of ART care services and the number of PLHIV receiving services at the facility. The health facilities involved were from Harari and Somali Regional State, Dire Dawa City, and the East and West Hararghe zones of the Oromia region. Nearly 80% of PLHIV in the research catchment areas received ART services from the health facilities engaged in this study. In Ethiopia, a comprehensive package for HIV care is provided free of charge. Clinicians and nurses provide clinical care, whereas trained counselors and outreach adherence supporters provide counseling and adherence support. In eastern Ethiopia, a viral load test service is provided at the Harari Health Research and Regional Laboratory and the Dire Dawa Regional Laboratory.

Study Design and Population
A multi-center, health facility-based cohort of PLHIV follow-up study was conducted in eastern Ethiopia. The cohort comprised all newly diagnosed HIV-positive clients who were on DTG-based first-line ART as per the current national guidelines. During the base line data collection period, study participants were enrolled consecutively and followed up on the ART for the next six months. All the study participants received standard HIV care at participating facilities, including initial ART adherence and routine laboratory monitoring. In addition, available clinical and immunological data at ART initiation were collected. Participants provided blood samples for the baseline VL test at the time of ART initiation and then at six months, as indicated in the national guidelines. Eligible individ uals who had already started ART prior to providing a blood sample and those who were critically ill at the enrollment period were excluded from the study.

Sample Size and Sampling Procedure
The sample size was calculated using the WHO sample size determination criterion for HIV pretreatment drug resistance (PDR) [27]. This study was part of a PDR mutation study among newly diagnosed HIV-positive individuals. All study participants enrolled for PDR determination were included upon informed consent and then assessed for thei status of virological suppression. At baseline, 235 of 252 (93.25% response rate) newly di agnosed HIV-positive individuals initiating DTG-based first-line ART were included in the cohort. A consecutive sampling method was applied to enroll HIV-positive clients a the selected health facilities, and the proportional distribution of study participants among the facilities was maintained based on the number of parents.

Data and Sample Collection
At baseline, a structured interviewer-administered questionnaire and checklists were used to collect socio-demographic factors, clinical data, laboratory parameters, and infor mation regarding the initiated first-line ART regimen. Study participants or caregiv

Study Design and Population
A multi-center, health facility-based cohort of PLHIV follow-up study was conducted in eastern Ethiopia. The cohort comprised all newly diagnosed HIV-positive clients who were on DTG-based first-line ART as per the current national guidelines. During the baseline data collection period, study participants were enrolled consecutively and followed up on the ART for the next six months. All the study participants received standard HIV care at participating facilities, including initial ART adherence and routine laboratory monitoring. In addition, available clinical and immunological data at ART initiation were collected. Participants provided blood samples for the baseline VL test at the time of ART initiation and then at six months, as indicated in the national guidelines. Eligible individuals who had already started ART prior to providing a blood sample and those who were critically ill at the enrollment period were excluded from the study.

Sample Size and Sampling Procedure
The sample size was calculated using the WHO sample size determination criterion for HIV pretreatment drug resistance (PDR) [27]. This study was part of a PDR mutation study among newly diagnosed HIV-positive individuals. All study participants enrolled for PDR determination were included upon informed consent and then assessed for their status of virological suppression. At baseline, 235 of 252 (93.25% response rate) newly diagnosed HIV-positive individuals initiating DTG-based first-line ART were included in the cohort. A consecutive sampling method was applied to enroll HIV-positive clients at the selected health facilities, and the proportional distribution of study participants among the facilities was maintained based on the number of parents.

Data and Sample Collection
At baseline, a structured interviewer-administered questionnaire and checklists were used to collect socio-demographic factors, clinical data, laboratory parameters, and information regarding the initiated first-line ART regimen. Study participants or caregivers/guardians of children/adolescents aged less than 18 years were interviewed by trained nurses using a face-to-face interview technique. Moreover, following standard operational procedures, approximately 5 mL venous whole blood samples were collected from all newly diagnosed HIV-positive individuals using tubes containing the anticoagulant ethylene diamine tetra-acetic acid. Plasma samples were harvested by centrifugation of whole blood at 3500 rpm for 5 min and aliquoted into Cryo tubes of 1.8 mL volume. Plasma samples were labelled with the medical registration number (MRN), before the format with some basic information to identify samples was sent to Harari Health Research and Regional Laboratory, Harar, Ethiopia, for HIV-1 RNA VL testing. The samples were then stored at −80 • C for long-term analysis. A similar approach to the baseline data collection method was used at the six-month follow-up. The interviews were conducted, data were collected, and relevant parameters were recorded using checklists. The VL was determined at six months as part of the routine follow-up.

Plasma Viral Load Determination
The collected plasma samples were processed and the VL tests were performed at the Harari Health Research and Regional Laboratory in Harar, Ethiopia. The samples were thawed at room temperature before the VL was run. The Abbott m2000sp automated sample preparation system and the Abbott m2000rt with quantitative Abbott RealTime HIV-1 assay (RT-PCR) (Abbott Molecular Inc., Des Plaines, IL, USA) were used for the extraction of HIV-1 RNA and the determination of plasma VL, respectively. The sample preparation (m2000sp) instrument was used for automated extraction, purification, and preparation of HIV-1 RNA. The m2000rt amplifies, detects, and measures the HIV-1 RNA load. A volume of 0.2 mL of the plasma samples was used for RNA extraction according to the manufacturer's instructions. The extracted HIV-1 RNA (eluent) was amplified and detected on the m2000rt Abbott platform, both at baseline and at six months of follow-up following the manufacturer's instructions (https://www.abbottmolecular.com accessed on 8 March 2023). The presence of the HIV-1 target sequence is indicated by the fluorescent signal generated using fluorescently labeled oligonucleotide probes on the Abbott m2000rt machine. The test was performed in the presence of positive, negative, and internal controls. The detection limit of the assay for the plasma sample was over the range of 40-10,000,000 copies/mL.

Data Analysis
EpiData Manager version 4.6.0.4 was used to code and enter the data. STATA/SE version 14.0 was used for the statistical analysis. For analysis, the log10 transformation of the HIV-RNA VL was used. Demographic and clinical characteristics were summarized using a descriptive analysis. Bivariate and multivariate logistic regression models were used to determine baseline factors associated with virological non-suppression at six months. All variables with p-value ≤ 0.25 from bivariate analyses were taken into the multivariable logistic regression analysis to investigate factors independently associated with virological non-suppression. A confidence interval (CI) of 95% and a p-value of <0.05 were considered statistically significant.

Socio-Demographics and Related Characteristics
In this study, 235 newly diagnosed PLHIV who initiated first-line ART were enrolled, with 70.6% of them female. The mean age of the participants was 33.9 years (SD: ±12.1), with a range of 2-70 years. The overall history of substance use at baseline was 35.3%, and the khat chewing habitual was 34% (80/235) (Table 1).

Baseline Clinical, Laboratory, and ART Profiles
Three-quarters of the study participants were from hospitals (74.9%). More than half of the newly diagnosed participants (60.4%) were linked to ART care through outpatient department (OPD) services. The current TB co-infection was 17%, whereas the overall comorbidity rate at the start of ART was 39.2% (Table 2).
Approximately 72.8% started ART treatment on the same day as diagnosis. At enrollment, 55.7%, 79.6%, 76.2%, 96.2%, and 94.9% of the participants were at WHO clinical stage I, working in functionality status, had a detectable VL (>150 copies/mL), had initiated TDF + 3TC + DTG (1J) first-line ART, and 2NRTI + INSTI classes, respectively. The proportion of participants with baseline HIV RNA VL results greater than 100,000 copies/mL was 23.4%, with results ranging from the target not detected to 4,102,070 copies/mL ( Table 2).

Treatment Outcomes and Virological Suppression Status at Six Months
The overall ART treatment outcomes at six months were 68.5% retention in ART, 16.6% loss to follow-up (LTFU), 8.9% transferred out, and 5.9% deceased (Table 3). Of the 161 patients retained on ART and available for the analysis, 8.7% (95% CI: 5.2-14.2) failed to achieve virological suppression at six months ( Table 3). The median HIV RNA VL among cases with virological non-suppression was 23,301 copies/mL (range: 3252-337,836 copies/mL). The TDF + 3TC + DTG first-line ART regimen was initiated in approximately 85.7% (12/14) of newly diagnosed HIV-positive individuals with virological non-suppression at six months ( Figure 2).

Baseline Factors Associated with Virological Non-Suppression at Six Months
Bivariate analysis showed that age ≤ 30 years, WHO clinical stage II and above, a history of substance use, and an HIV RNA VL greater than 4-log10 copies/mL were baseline factors associated with virological non-suppression at six months (Table 4). There was

Baseline Factors Associated with Virological Non-Suppression at Six Months
Bivariate analysis showed that age ≤ 30 years, WHO clinical stage II and above, a history of substance use, and an HIV RNA VL greater than 4-log10 copies/mL were baseline factors associated with virological non-suppression at six months (Table 4). There was a statistically significant difference in baseline mean VL for PLHIV-achieved viral suppression and non-suppression at six months (140,717.7 vs. 628,569.6 copies/mL; t = −3.0070, p = 0.0032). To control for confounding factors, variables with p-values of 0.25 in bivariate analysis were imported into a multivariate logistic regression model (Table 4). Multivariate logistic regression analysis revealed that age ≤ 30 years at ART initiation (AOR = 8.90, 95% CI: 1.85, 42.77), a history of substance use at baseline (AOR = 7.50, 95% CI: 1.41, 39.97), and a baseline HIV RNA VL greater than 4log10 copies/mL (AOR = 12.64, 95% CI: 1.65, 96.48) were independently associated with virological non-suppression at six months. The odds of virological non-suppression at six months were approximately nine times (AOR = 8.90) higher in those younger than 30 years old compared to those older than 30 years old. Moreover, among PLHIV with a history of substance use and a VL greater than 4-log10 copies/mL at baseline, the likelihood of viral non-suppression at six months was 7.5 (AOR = 7.50) and 12.6 (AOR = 12.64) folds higher, respectively (Table 4).

Discussion
The magnitude of virological non-suppression in this study was 8.7%, and the predictors of virological non-suppression at six months were age less than 30 years, a history of substance use, and a baseline VL > 4-log10 copies/mL. Virological suppression is an important factor in PLHIV health maintenance and plays a great role in the prevention of new HIV cases. In contrast, non-suppression of virological treatment is a key challenge for HIV programs, particularly in LMICs, because the likelihood of drug resistance and subsequent viral transmission, especially that of drug-resistant strains, increases when virological suppression is not achieved.
The rate of virological non-suppression was comparable with studies conducted in various places in Ethiopia: 8.3% at TASH [28], 10.24% in the North Shoa Zone [29], 11.8% in Gondar [30], 12.2% at Delgi Hospital [10], 12% in southern Ethiopia [31], and 10.5% in a study conducted in Dar Es Salaam, Tanzania [32]. Virological non-suppression, however, was lower than that reported from Kenya (24%) [33] and Cameroon (23.2%) [34]. The disparity might be attributed to differences in the study populations, ART follow-up period, advances in ART classes and regimen, study design, the cut-off points to define the virological non-suppression, and adherence [12,25,30]. Additionally, the inconsistency may also be due to the type of interventions and updates in HIV care and treatment guidelines which were periodically optimized to HIV-positive patients toward ART treatments and the type of ARV regimen given up on their routine follow-up [35].
In the current cohort, viral non-suppression was unlikely among PLHIV aged less than 30 years compared to their counterparts. The result was similar with other studies from SSA African countries such as Uganda, Tanzania, and South Africa [36][37][38]. The ART regimen provided to younger HIV-positive people may help explain this. For younger children, a protease inhibitor-based (LPV/r-based) regimen is recommended, though EFV-based regimens can be prescribed. This may affect the virological suppression in the long-term because a high level of drug resistance was already reported in EFV-based regimens [21,39,40]. If the dose is not adjusted with weight increase, the concentration of the drug will be suboptimal, and viral replication cannot be controlled. Weight gain was commonly reported among PLHIV using ART in the dolutegravir era [41][42][43]. Moreover, young individuals have difficulty achieving acceptable ART adherence [38].
This study showed that the likelihood of virological non-suppression was 7.5 folds higher among PLHIV who reported substance use (khat chewing, alcohol drinking, cigarette smoking, etc.) during ART initiation. Studies conducted in South Africa revealed that substance use had higher odds of unsuppressed viral load than those who reported no substance use [44,45]. Moreover, at baseline, stimulant use was associated with inconsistent viral suppression status. This was because substance use was associated with poor adherence. These results highlight the importance of considering substance use in the context of viral suppression [44].
The current study also found that PLHIV with a baseline VL > 4-log10 copies/mL had higher odds of virological non-suppression six months after first-line ART. Previous studies found that a low baseline VL was a predictor of viral suppression (AHR = 1.56) [46], whereas a higher VL at treatment initiation might have a larger HIV reservoir, which might require a longer time to achieve VL suppression than for those with a low VL [47]. Furthermore, studies have shown that HIV-positive individuals with a baseline plasma VL > 100,000 copies/mL were more likely to fail viral suppression regardless of ARV regimen receiving, including integrase inhibitor-based cART regimens (versus < 100,000 copies/mL) [23,48,49]. In our study, nearly a quarter (23.4%) of the participants had a baseline VL > 100,000 copies/mL during treatment initiation. Therefore, there is an increased risk of HIV transmission to partners when PLHIV carries a higher VL for a longer time after ART initiation. This can provide insights into the interventions that are necessary to enhance health outcomes and mitigate the onward transmission of HIV.
In summary, baseline VL testing is not performed in the majority of LMICs, including Ethiopia, and people who test positive for HIV begin ART without a VL test. In light of this, in addition to the efforts to achieve the targets of the 95s, it is preferable to reconsider the current guidelines for the routine and the first VL test at six months for PLHIV. Not only can baseline VL testing benefit HIV-positive individuals, but it can also assist healthcare professionals in implementing VL-triggered adherence support strategies that may improve patient treatment results [50]. As a result, taking the initiative for baseline VL testing for HIV-positive clients starting ART may be required to properly monitor ART adherence from the start and optimize the benefits of HIV care and treatment programs. Furthermore, cost-effective, affordable, rapid, and decentralized VL testing might increase the uptake of baseline VL and viral suppression in general [26,50,51]. Moreover, considering pretreatment HIV drug resistance genotypic testing at baseline could be helpful in identifying patients at a higher risk of virological failure and contribute to the long-term success of viral suppression [52]. During the inception of ART, focused interventions are also necessary, such as paying attention to younger HIV-positive individuals [38] and those who have substance use habits.
This study has several strengths, including being conducted at multiple sites over a period of six months in eastern Ethiopia. The cohort comprised newly diagnosed PLHIV who started ART in routine HIV care. Baseline factors that can predict virological nonsuppression were assessed, including baseline VL testing and the untapped area in Ethiopia. This study provides insight by generating baseline regional epidemiological and clinical data for the health sectors, which will be used for program-wide trend monitoring.
However, this study has some limitations. The result of HIV drug resistance was not included, which could lead to virological non-suppression. The rate of virological non-suppression may be underestimated owing to the relatively small sample size because of the loss to follow-up, transfer out, and deceased participants. Furthermore, those with low baseline viremia at enrollment could deceitfully lower the virological non-suppression at six months. In this study, COVID-19 may have also affected the adherence of the patients. Moreover, immuno-hematological parameters were not adequately captured, as they were not implemented routinely because of their minimal importance and shortage of supplies. Therefore, it is important to consider these limitations when interpreting the conclusions of this study.

Conclusions
Virological non-suppression after six months of receiving DTG-based first-line ART is found to be optimal in newly diagnosed HIV-positive people. A younger age, a history of substance use, and a VL more than 4-log10 copies/mL at ART inception were baseline predictors of virological non-suppression at six months. Therefore, HIV treatment and care programs, as well as stakeholders, should pay due attention to PLHIV with the identified determinants-potentially through targeted interventions such as optimizing differentiated service delivery for those who did not achieve viral suppression. This finding emphasizes the significance of baseline VL monitoring. To improve treatment outcomes and reduce the incidence of HIV transmission, efforts are needed to introduce cost-effective, rapid, and decentralized baseline VL testing. Furthermore, conducting more comprehensive and broader research is recommended to explore reasons for virological non-suppression in the study area. The study was conducted in accordance with the Declaration of Helsinki. Moreover, the laboratory results of patients with failed virological suppression at six months were reported to health care providers to enhance adherence counseling and to receive support as per the guidelines.
Informed Consent Statement: Informed consent was obtained from all study participants involved in this study. Written informed consent and assent (in the case of adolescents) was obtained from each study participant and the study was conducted in an anonymous manner.

Data Availability Statement:
The data presented in this study are available on reasonable request from the corresponding author. The data are not publicly available due to privacy.