Immune recovery after antiretroviral therapy initiation: a challenge for people living with HIV in Brazil

Immune recovery reflects health conditions. Our goal was to estimate the time it takes to achieve immune recovery and its associated factors, in people living with HIV (PLHIV), after antiretroviral therapy (ART) initiation. A historical cohort study was performed among PLHIV (> 18 years-old) in Minas Gerais State, Brazil, using data from healthcare databases. Patients initiating ART between 2009-2018, with T-CD4+ lymphocytes and viral load recorded before and after antiretroviral therapy were included. The outcome is achievement of immune recovery, defined as the first T-CD4+ > 500 cells/µL after ART initiation. Explanatory variables were age, gender, place of residence, year of ART initiation, baseline viral load and T-CD4+, viral load status, and adherence to ART at follow-up. Descriptive analysis, cumulative, and person-time incidences of immune recovery were estimated. Median-time to immune recovery was estimated using Kaplan-Meier method. Factors associated with immune recovery were assessed by Cox regression. Among 26,430 PLHIV, 8,014 (30%) were eligible. Most were male (67%), mean age 38.7 years, resided in non-central region, median-baseline T-CD4+ = 228 cells/µL (< 200 cells/µL


Introduction
Antiretroviral therapy (ART) has been shown to be effective in reducing HIV viral load and achieving immune recovery (optimal T-CD4+ lymphocytes count) 1,2 . Viral load suppression and immune recovery are the first goals when initiating treatment. Viral load suppression is identified by RNA plasma level and defined according to the detection limit of commercially available testing 2 . In contrast, the interval of normal levels of T-CD4+ ranges widely from 500 to 1,500 cells/µL 3 . Thus, there is no consensus on how to best identify immune recovery. Some studies focus on a specific T-CD4+ threshold based on guidelines for ART initiation (e.g. 200, 350 or 500 cells/µL), whereas others focus on the rate of increase of T-CD4+ over time (i.e., the T-CD4+ slope) 4 .
After ART initiation, the time needed to achieve viral load suppression is typically measured in months, while immune recovery may take years. The median time for viral load suppression ranges from 3.4 months to 4.5 months regardless of the ART regime 5,6 . In contrast, immune recovery is a gradual process which takes place over a long period of time. The initial reconstitution of T-CD4+ occurs up to 6 months, from the beginning of treatment. In a second phase, the increase in T-CD4+ counts is slower and lasts up to 2 years following treatment initiation. Finally, the last and slowest phase of recovery can last 7 years or more 2 .
Multifactorial determinants may delay, or prevent, immune recovery even after effective response to ART; and the mechanisms that lead to discordant immune response are not well understood 4,7 .
Older age, persistent immune activation, low level of continuous HIV replication, coinfections, and low pre-therapeutic and lower T-CD4+ counts after ART initiation may predispose individuals to have poor immune recovery 7,8,9 . Additionally, some studies have shown that antiretroviral agents -such as zidovudine -may be toxic for hematopoietic progenitor cells 2 , further delaying immune recovery. Other studies have reported a better immunological response with the use of integrase inhibitor 10 .
Immune recovery is an indicator of improvement in the health in those who seek treatment. An impaired immunological response is linked to increased risk of disease progression and even death; managing immune failure is complex 2,11 . People living with HIV (PLHIV) who experience immune recovery through ART have mortality rates close to HIV-negative individuals of same age and gender 12 . A study of 80,642 HIV patients from 31 European countries who initiated ART between 1998 and 2008 found a similar mortality rate in heterosexuals with HIV, who maintained T-CD4+ counts > 500 cells/µL over 5 years 12 . A systematic review showed that the risk of mortality varied from 1% to 7% (median follow-up 3.7 years) for patients who attained immune recovery, and from 3% to 23% (median follow-up 2 years) for those who failed to attain immune recovery 7 .
Recently, notable improvements in antiretroviral therapy's ability to suppress the viral load has resulted in an increase of PLHIV who are undergoing treatment, consequently reducing the transmission of HIV 5 . There has been a growing interest in understanding the prognosis of those who undergo ART, and the risks of undesirable long-term outcomes. Immune recovery is a critical component to understanding the health status and predicting the occurrence of chronic disorders in PLHIV. The aim of this study is to estimate the time it takes to achieve immune recovery, and investigate potential contributing factors, in a cohort of PLHIV, who have initiated ART from 2009 through to 2018, in Minas Gerais State, Brazil.

Study design and population
A historical cohort study was carried out within an open cohort of PLHIV who began ART between January 1, 2009, and December 31, 2018, in Minas Gerais State. HIV infected individuals aged 18 years or older, with baseline T-CD4+ ≤ 500 cells/µL, and with documented viral load and T-CD4+ counts (before and after ART initiation) were eligible for analysis. The study was approved by the Ethics Research Committee of the Federal University of Minas Gerais (CAAE 627.10316.8.0000.5149).

Data source and outcome
Laboratory data (viral load and T-CD4+ measurements), therapeutic variables (ART use and adherence), and demographic variables (age, gender, and place of residence) were obtained from two national healthcare databases of the Brazilian Unified National Health System (SUS): Medication Logistics Control System (SICLOM) and Laboratory Test Control System (SISCEL), required nationwide for ART dispensing and laboratory results, respectively 13 . Both, SICLOM and SISCEL, had the same identification number for each patient, which allowed the linkage of the databases.
ART is provided free of charge to all PLHIV in Brazil, through public healthcare facilities. Thus, ART dispensing data covers PLHIV accessing public and private health care. SISCEL covers mainly (but not exclusively) PLHIV from public health care 13 .
Our outcome of interest was the attainment of immune recovery, defined by the first T-CD4+ count > 500 cells/µL (the first limit of T-CD4+ normal level) observed during follow-up 14 , among those with initial T-CD4+ count < 500 cells/µL. Follow-up time was defined as the period between the date of the first ART prescription and the date of the first occurrence of T-CD4+ counts > 500 cells/µL (outcome) or the last date at which T-CD4+ counts < 500 cells/µL was recorded (censoring). The date of ART initiation was based on the first date of treatment dispensing, after checking that there was no other record of ART in the previous three years.

Baseline variables
Baseline variables, collected at ART initiation date were age, gender, place of residence (central or non-central region within Minas Gerais State), year of ART initiation (before or after 2013), viral load measurements, and T-CD4+ counts. Viral load measurements (log 10 scale) and T-CD4+ counts were obtained within 3 months prior to ART initiation. The central region, where the state capital is located, comprised approximately 28% of the state population (5,384,904 inhabitants), while the population of the non-central region was estimated at 15,783,887 inhabitants, in 2019 15 . The year 2013 was chosen as the threshold for ART initiation due to the change in national HIV recommendation guidelines to early treatment initiation concerning the T-CD4+ counts or clinical stage 16,17 .

Data management and time-varying variables
Viral load status and ART adherence were time-varying variables evaluated during the follow-up period. Each T-CD4+ segment -period between two consecutive dates of T-CD4+ count of the same individual in the follow-up period -is associated with a time-updated viral load status and ART adherence assessed within the segment.
Viral load status was classified as undetectable (< 1.69 log 10 copies/mL), detectable (> 1.69 log 10 copies/mL), or absent (no measurement) 18 . When there was more than one viral load result within the same T-CD4+ segment, the last measurement was considered. The mean time of ART dispensing within each T-CD4+ segment was used as proxy for treatment adherence 19 . It was calculated by dividing the duration, in days, of each T-CD4+ segment by the number of registered ART prescriptions dispensed within the corresponding segment. Intervals < 45 days were classified as ART adherence and intervals > 45 days were classified as absence of ART adherence. If no ART was dispensed within the T-CD4+ segment, the individual was considered adherent if the final date of the segment fell within 45 days of the last ART dispensing.

Data analysis
Descriptive analysis was performed to characterize the study's population. Absolute and relative frequencies, mean, standard deviation (SD), median, and inter-quartile range (IQR) were calculated. Cumulative and person-time incidences of immune recovery were estimated. Cumulative incidence of immune recovery was calculated by dividing the number of individuals who achieved immune recovery by the total of individuals at baseline. Person-time incidence of immune recovery was calculated dividing the number of individuals who achieved immune recovery by the total person-time Cad. Saúde Pública 2021; 37(9):e00143520 of all individuals during the follow-up, corresponding to the period between the data of the first ART prescription and the date of the immune recovery occurrence (outcome) or the last date of T-CD4+ counts < 500 cells/µL (censoring).
Overall median time to immune recovery and time to immune recovery stratified by baseline variables were estimated by the Kaplan-Meier method with 95% confidence intervals (95%CI). For time-varying variables (ART adherence and viral load status), stratified Kaplan-Meier curves were calculated based on the information recorded in the first T-CD4+ segment of follow-up 20 .
The association between potential explanatory variables and immune recovery attainment was assessed by Cox proportional hazard regression models 21 . Univariate analyses were conducted, followed by multivariate modeling. The final model was selected by the likelihood ratio test 21 . The magnitude of the associations was estimated using adjusted hazard ratio (HR), with 95%CI. The assumption of proportionality of the hazard was assessed by Schoenfeld residual analysis 21 . Database management and statistical analyses were performed using the Statistical Analysis System (SAS), version 9.4 (https://www.sas.com/), and R Software, version 3.0.1 (http://www.r-project.org), respectively. Baseline characteristics are presented in Table 1. Approximately 50% of the study population initiated ART before 2013, 67% (n = 5,365) were male, had mean (SD) age of 38.7 (11.5) years old, and 4,270 (53%) lived in non-central region. The median (IQR) and the mean (SD) baseline T-CD4+ was 237 (108-334) cells/µL and 228 (137) cells/µL, respectively, and 43% had T-CD4+ counts < 200 cells/µL. The median (IQR) viral load was 4.7 (4.2-5.3) log 10 copies/mL and approximately 99% of the individuals presented detectable viral load (> 1.69 log 10 copies/mL).

Follow-up and median time to immune recovery
The median (IQR) duration of follow-up was 14.0 (5.4-32.2) months while the total follow-up time was 15,874 person-years. The median (IQR) number of T-CD4+ segments within the follow-up was 2 (1-5). The cumulative incidence of immune recovery was 58% (95%CI: 57-58) (n = 4,678), corresponding to an incidence rate of 29.47/100 person-years. The median time to immune recovery was estimated at 22.8 months (95%CI: 21.9-24.0). Those who presented a shorter median time to immune recovery were female, less than 38 years of age, living in central region; they had initiated ART after 2013, baseline T-CD4+ count > 350 cells/µL, detectable viral load at baseline, and undetectable viral load and ART adherence during follow-up (Table 2).
Baseline T-CD4+ counts and ART therapy adherence measured at follow-up showed a considerable effect on the median time to immune recovery. Participants with T-CD4+ counts > 350 cells/µL at ART initiation achieved immune recovery, approximately, 10.5 times faster than individuals with T-CD4+ counts < 200 cells/µL: median time = 5.8 months and 60.7 months, respectively (Table 2; Figure 2). Similarly, ART adherence during follow-up decreased the median time to immune recovery more than 7-fold: median time = 7.7 months compared to 57.6 months in ART no adherence (Table 2; Figure 2). Slight differences in the Kaplan-Meier curves were found for gender, age, place of residence and year of ART initiation.

Univariate and multivariate Cox regression analyses
All covariables were statistically significant (p < 0.05) predictive of time to immune recovery in the univariate analysis and were therefore included in the multivariate modeling. To obtain a parsimonious model, period of ART initiation and place of residence were removed from the final model. Thus, age, gender, baseline T-CD4+ counts, baseline viral load, ART adherence, and viral load at follow-up remained in the final multivariate model (Table 3). Women, 38 years-old and younger, were less associated with immune recovery: HR = 1.12  (Table 3). The final model explained 77% of the variability in outcomes, Schoenfeld residual analysis indicated adequate proportionality of risks for gender and viral load baseline.

Discussion
Our study estimated the median time needed for immune recovery for PLHIV, from a large Brazilian state, undergoing ART, and assessed the potential factors, such as: age, gender, year of ART initiation, place of residence, T-CD4+ counts, viral load measurements and treatment adherence. During the study period, Brazilian HIV guidelines were updated concerning the optimal threshold of baseline T-CD4+ to initiate ART. Since 2013, the T-CD4+ threshold increased from 350 to 500 cells/µL and, after 2017, the recommendation was to make ART available to all PLHIV, regardless of T-CD4+ counts 17,22 . Despite these dynamic updates, our study indicated that a considerable proportion of individuals had late ART initiation over the study period: 56% and 44% of the participants initiated treatment with T-CD4+ counts less than 200 cells/µL before and after 2013, respectively. Similar results were observed in a large study in South Africa. In 844,699 HIV individuals initiating ART between January 2010, and December 2014, the study estimated the proportion of baseline T-CD4+ < 200 cells/µL varying from 58% to 36% 23 .
A high proportion (42%) of individuals did not achieve immune recovery in our study, despite the long period of follow-up. Our findings are similar to a study in the South Africa, in which 55.6% (n = 469,825) of HIV participants did not achieve immune recovery and the median time (IQR) to immune recovery was estimated to be 17.3 (10.0-29.6) months 23 . Since there is a higher risk of AIDS, Cad. Saúde Pública 2021; 37(9):e00143520 non-AIDS diseases, and even death occurring in individuals with poor immune recovery, they require continued T-CD4+ monitoring in order to ensure effective management of immune failure 24,25 . The U.S. Office of AIDS Research Advisory Council (OARAC) have maintained the recommendation that T-CD4+ counts should be monitored every 3 to 6 months during the first 2 years of ART initiation, and every 12 months after 2 years of treatment 26 . However, several studies have suggested that T-CD4+ monitoring among virologically suppressed individuals may not be necessary, and the reduction in T-CD4+ tests may free up resources to put more people on ART 27 . Nevertheless, most of these studies are randomized trials, which include a reduced sample of patients with higher T-CD4+ counts before ART initiation and immune recovery defined by reduced T-CD4+ threshold of 200 cells/µL 28,29,30,31 , and thus reduced follow-up may not be advisable in real-world populations with a higher proportion of more immune-compromised patients.
Our study showed that the period of ART initiation, place of residence, gender, and undetectable viral load after ART initiation had a lower impact on the time to immune recovery. After 2013, evidence pointed to a decrease of the HIV transmission through early treatment; as a result, the recommendations concerning ART initiation were updated to immediate treatment after HIV diagnosis      33,34 . This could partially explain the borderline effect on the time to immune recovery according to regions. Concerning gender, there are several evidences indicating that time to achieve immune recovery is usually higher among men than women 8,35,36 . Although, our findings showed that the median time and HR to immune recovery was barely influenced by gender.
Cad. Saúde Pública 2021; 37(9):e00143520 We found that immune recovery was associated with detectable baseline viral load and undetectable viral load at follow-up. In fact, levels of T-CD4+ at ART initiation and their changes over the course of ART-treated HIV infection (T-CD4+ slope) are the most important predictors of immune recovery achievement 24,25 and viral load measurements may indirectly contribute to successful immune recovery. The literature shows that detectable viral load at ART initiation requires closer viral load monitoring with more frequent measurements, which would improve ART adherence, leading to viral load suppression and immune recovery 5,37,38 .
Finally, higher T-CD4+ counts at the ART initiation and treatment adherence at follow-up were the strongest predictors of immune recovery occurrence. Late ART initiation and adherence have remained key challenges in HIV control. In Brazil, since 2016, approximately 26% of PLHIV have had a late treatment start 39 . Two cross-sectional studies, performed in 2010 (N = 1,970) and 2017 (N = 356) in Brazilian Northeast, estimated a proportion of late ART initiation of 50% and 41%, respectively 40,41 . Some factors associated with late ART initiation comprised lower risk behaviors, probably due to a lack of perception of HIV infection risk: heterosexual couples with children, one sexual partner, no drug users, no history of sexually transmitted infection 40,41,42 . These results reinforce the need for HIV testing. Different measurement tools and ART adherence definitions used across different studies may limit comparisons. However, there is a broad consensus that ART adherence remains a challenge for HIV control worldwide 43 . In Brazil, non-adherence estimates ranged from 25% to 30% between 2015 and 2018 39 . A Brazilian cross-sectional study (N = 120) conducted between 2007 and 2014 showed a prevalence of non-adherence of 43.3% 44 . Missing medical appointments, mental health conditions, adverse effects of ART and use of illicit drugs are some risk factors associated with non-adherence to treatment 43,45,46 . On the other hand, the presence of AIDS diagnosis, which requires more frequent T-CD4+ measurements, improves adherence to ART and also, as a result, immune recovery monitoring. Thus, male, older patients, with low T-CD4+ counts and undetectable viral load at ART initiation, presenting non-adherence to ART and detectable viral load during treatment should be prioritized for T-CD4+ monitoring in order to ensure immune recovery.
Some limitations of our study must be addressed. First, the sample was remarkably reduced from the total population of PLHIV due to the unavailability of T-CD4+ and viral load results before and after the start of ART. A large proportion of missing data were probably due to individuals receiving healthcare in Minas Gerais state (27%), including T-CD4+ counts and viral load monitoring, at private services (Brazilian Health Informatics Department. http://tabnet.datasus.gov.br/cgi/tabcgi. exe?idb2012/f16.def, accessed on 21/Sep/2020), whose data have not to be compulsorily recorded in the SUS databases. Despite the recognized importance of T-CD4+ count in evaluating disease status at baseline, it is possible that some patients, especially those who were clinically stable, had initiated ART without performing T-CD4+ count in view of the recommendations of early treatment initiation since 2013 17 . Additionally, the shift to viral load monitoring in ART could explain, partially, rates of missing T-CD4+ count in the follow-up period. Lastly, the increased demand for laboratory monitoring, generated from the early ART initiation, can be a challenge for low-and middle-income countries in providing adequate coverage of both T-CD4+ count and viral load tests. In an African study aimed at predicting T-CD4+ count recovery, the number of participants was reduced by 83% (from 3,981,104 to 1,070,900) mainly due to the missing data on T-CD4+ lymphocytes exams 8 . National studies of Melchior et al. 47 evaluated the quality of HIV public healthcare in seven Brazilian States between 2000 and 2001 (N = 92,400 patients: 72% of all Brazilian HIV-patients); they showed that T-CD4+ counts and viral load measurements were available in the majority of services, and yet approximately 50% did not perform those exams. Currently, there is no known national information regarding this issue. Thus, monitoring the availability and frequency of T-CD4+ count performance may allow realistic and ongoing assessment of immune recovery over time.
Despite the reduction in sample size, it was possible to conduct a longitudinal analysis from available data of a considerable number of PLHIV, providing valuable information for HIV/AIDS healthcare. Further, selecting participants with lower baseline T-CD4+ counts may have led our results to underestimate the frequency of immune recovery. These participants, including those with late diagnosis/treatment and with co-infections (e.g. tuberculosis, hepatitis B and C), are more likely to test routinely and to achieve immune recovery less frequently 48 . In fact, the mean (SD) of baseline Cad. Saúde Pública 2021; 37(9):e00143520 T-CD4+ counts of population excluded (prevalent cases) was 674 (134) cells/µL versus 228 (137) cells/µL of selected population of the study. The last limitation refers to the PLHIV who initiated ART in the latter years of the study period and therefore had limited follow-up time, with unknown contribution to our results.
This study points to the value of using large, linked public databases for following HIV treatment over time. The SISCEL and SICLOM databases were initially created for the management of laboratory results and dispensing of antiretrovirals, however, they have become a widely used source for research purposes. Joining the data from SISCEL and SICLOM with other databases such as Brazilian Information System for Notificable Diseases (SINAN) or Mortality Information System (SIM) represents an important field of research that could assist in clarifying crucial issues concerning the disorders occurred in PLHIV under long-term antiretroviral therapy but with suboptimal immune recovery. For subsequent studies, these data could also be used to verify the effectiveness of recently adopted antiretrovirals, such as dolutegravir, by assessing viral suppression and achievement of immune recovery.

Conclusion
Our study highlights the importance of early ART initiation, ART adherence and T-CD4+ counts in the attainment of immune recovery. Nevertheless, there is a significant proportion of PLHIV with late ART initiation, which is not in line with the current guidelines of reducing the frequency of T-CD4 counts tests 49 . Thus, interventions to ensure that people living with HIV will be immunologically monitored, besides earlier HIV diagnosis and treatment, represent the main challenges to achieve immune recovery in this Brazilian State.