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Determinants of immunological recovery following HAART among severely immunosuppressed patients at enrolment to care in Northern Ethiopia: a retrospective study

Objective This study aimed to identify determinants of immunological recovery following highly active antiretroviral therapy (HAART) among severely immunosuppressed patients at enrolment to care in Northern Ethiopia. Methods A retrospective study. Setting The study was done in Tigray Region, Northern Ethiopia. Participants The study was done among severely immunosuppressed (<200 CD4 cells/mm3) individuals at initial enrolment to care and whose samples were sent for viral load determination from April 2015 to March 2019 in Tigray Health Research Institute. Main outcomes The main outcome variable was immunological recovery, modelled using binary logistic regression. Results Among the 9687 patients with severe immunosuppression at enrolment, 2746 (28.35%, 95% CI 27.45% to 29.26%) had immunological recovery following HAART for at least 6 months. Male gender (adjusted OR (AOR)=0.50, p<0.001), age 20–34 years old (AOR=0.33, p<0.001), age ≥50 years old (AOR=0.26, p<0.001), WHO clinical stage III (OR=0.68, p=0.036) and viral non-suppression (AOR=0.38, p<0.001) were strong predictors of immunological failure. Conclusions Immunological recovery following HAART was low among severely immunosuppressed individuals at enrolment to care. Gender, age, WHO stage III and viral non-suppression were determinants of immunological recovery. Male patients, adolescents and virally non-suppressed patients should be identified as groups at higher risk for immunological failure. Therefore, greater support and intensive counselling should be prioritised among adolescents, men and virally non-suppressed patients for better immunological recovery.


INTRODUCTION
HIV infects CD4+ T cells, replicates in these immune cells and gradually depletes these immune cells, mainly CD4 lymphocytes, which are key in cell-mediated immune response, thereby contributing to poor humoral response against invading pathogens. [1][2][3][4] After progressive depletion of CD4+ T cells, the risk of opportunistic infections increases. Nevertheless, initiating antiretroviral therapy (ART) can help achieve suppression of viral replication, leading to an increase in CD4+ T cell counts and restoring the body's capability to fight against opportunistic infections. 1 5-9 This results in immune function recovery, accompanied by a dramatic decrement in morbidity and AIDS-related mortality. 10 11 Globally, 20.9 million HIV-infected people were estimated to have received ART by June 2017. 12 Strengths and limitations of this study ► The study was done on a relatively higher sample size, which can be generalised to the national level. ► The study was done using routinely collected public health programme data which can reflect the outcome and effectiveness of current antiretroviral therapy (ART) in severely immunosuppressed patients. ► Appropriate statistical methods were used to present the findings of the study. ► There were no data available when baseline and recent CD4 T cell counts were determined and hence the time of immunological recovery is not known. ► Due to the nature of the secondary data, the analysis misses some important variables, such as the existence of coinfection, drug side effects during followup, body mass index and grade of ART experience among HIV-infected patients.

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The WHO recommends (as of 2015) that HIVinfected adults should receive ART right away irrespective of their CD4 count. 13 After commencing ART, the expected outcome is either immunological recovery or failure. Immunological failure is defined as a CD4 count at or below 250 cells/mm 3 following clinical failure or persistent CD4 level below 100 cells/mm 3 in adults and adolescents. 14 The minimum normal CD4 count for the general adult population is 500 cells/mm 3 , and attaining this level after initiating ART is defined as immunological recovery. 14 15 There are a variety of factors that could influence the CD4 cell count of human beings. Studies have reported that individuals who start ART at a higher CD4 count (≥300 cells/mm 3 ) have their CD4 cell count return to nearly normal or normal (≥500 cells/mm 3 ) than those who start at a lower baseline (≤200 cells/mm 3 ). 14 16 Other different studies have also shown that poor immunological recovery and survival were observed in patients who initiated ART at low CD4 counts. [17][18][19] Moreover, patients with poor immune recovery have shown to have a higher risk of developing both AIDS-related and non-AIDSrelated clinical events. 14 Similar studies have also shown a link between viral suppression and improved immune response while on ART. [20][21][22] On the other hand, there is evidence that older people have poorer response to ART treatment. 23 24 Despite ART coverage expanding dramatically in Ethiopia, nothing is known about the determinants of immunological recovery. However, immunological failure was considered as a proxy marker for virological failure. 25 26 Thus, it influences the performance of the virological suppression goal of the Joint United Nations Programme on HIV/AIDS third 90-90-90 target, 27 which aims to achieve 90% virological success in patients on ART.
This study is part of a previously published study, the results of which have not been presented elsewhere. 28 None of the previously published studies identified the determinants of immunological recovery following highly active antiretroviral therapy (HAART) among severely immunosuppressed individuals at enrolment to care in Tigray Region. [28][29][30] Hence, it is important to identify determinants of immunological recovery among patients with <200 CD4 cells/mm 3 at enrolment to enhance immunological recovery and thereby survival. Therefore, this study aimed to identify determinants of immunological recovery following HAART among severely immunosuppressed patients at enrolment in Northern Ethiopia. This study will aid programme experts, leaders and clinicians in Ethiopia and other resource-limited settings in identifying determinants of immunological recovery following HAART among severely immunosuppressed patients at enrolment so as to reduce complications and enhance management of these patients.

Study setting
The study was done in Tigray Region, which is the sixth largest by surface area and the fourth most populous of the nine regional states of Ethiopia 31 (see figure 1).

Study design
A retrospective analysis study design was used.

Participants
The participants of this study were severely immunosuppressed patients at enrolment and on HAART for at least 6 months and whose samples were sent to Tigray Health Research Institute (THRI) for viral load (VL) determination through standard sample transportation technique from April 2015 to March 2019. THRI serves as a regional reference laboratory and is the only centre for VL determination in Tigray Region and for some parts of Afar Region. The study was done among 9687 patients with complete data on demographics, clinical and immunological characteristics, and VL in the THRI database.

Eligibility criteria
Inclusion criteria ► Patients enrolled in ART care for at least 6 months.
Exclusion criteria ► Individuals with ≥200 CD4 cells/mm 3 at enrolment to care. ► Subjects younger than 15 years.

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Sampling procedure To come up with the sample, all records of HIV in the THRI database with complete information were extracted. All individuals who fulfil the eligibility criteria were included in the study (see figure 2).

Data collection tools and procedures
All data in the database were exported to Microsoft Excel 2013 and data verification and filtration were done before exporting to STATA V.14.0. Laboratory testing methods included the following: RNA extraction and plasma VL determination were done from samples of plasma from each patient sent to THRI. HIV-1 RNA was extracted from 0.2 mL of plasma using Abbott m2000sp automated sample preparation system (Abbott Molecular, USA). Extracted RNA was measured using Abbott m2000rt quantitative real-time HIV-1 assay (Abbott Molecular) with HIV-1 RNA detection level of 40-10 million copies/ mL based on the manufacturer's procedures. The CD4 count was determined at respective healthcare facilities using FACSCount, and baseline and recent CD4 counts of each patient were sent to THRI through the VL standard referral form.

Patient and public involvement
The study involved immunosuppressed patients at enrolment following HAART in Tigray healthcare facilities.
Operational definitions ► Immunological recovery: patients enrolled to care whose CD4 count was below 200 cells/mm 3 at baseline and who have achieved ≥500 CD4 cells/mm 3

Data management and analysis
Analysis was done using STATA V.14.0 to estimate the proportion and determinants of immunological recovery among patients with severe immunosuppression at baseline (at enrolment to care) following HAART. The variable 'viral suppression' was categorised based on WHO categorisation for low-income and middle-income countries (LMICs). Missed values were filtered and excluded for all variables; hence, there was no issue on missing values in the final data set. The outcome variable was dichotomised (1=immunological recovery and 0=immunological non-recovery). Descriptive analysis was done for demographic, clinical, virological and immunological characteristics. Cross tabulations were done among the different variables with the outcome variable. The proportion of patients who had immunological recovery was obtained by dividing the total number of patients with severe immunosuppression who have achieved immunological recovery by the total number of patients with severe immunosuppression at baseline. The proportion of immunological recovery was further evaluated by age group, sex, adherence level, pregnancy status, breastfeeding status, WHO staging, regimen type, treatment line, reason for VL test, health facility ownership and health facility level.
Statistical significance was considered at p<0.05 (twosided) in all tests. Robust SE type was used in the analysis of bivariate and multivariate logistic regression. To identify determinant factors associated with immunological recovery, bivariate analysis was first conducted to identify the strength of association with immunological recovery at crude level. Crude OR was calculated at the bivariate logistic regression. All significant variables in bivariate analysis were entered into the multivariate analysis. Multivariate binary logistic regression was used to identify Open access factors independently associated with immunological recovery at adjusted level. As pregnancy and breastfeeding status apply to women, both variables were excluded from the final statistical modelling. Collinearity was omitted in statistical modelling of immunological recovery. Model adequacy was checked by Hosmer-Lemeshew test.

Background characteristics
Among the 9687 study subjects, about 61.34% were women. The median age of the patients was 40 (IQR 33-45). The age of most patients lies in the 35-39 and 40-44 years age groups, accounting for 21.75% and 21.08%, respectively. Around 95% of the patients were enrolled in governmental healthcare facilities.
Among the 5908 female patients, 34 (0.57%) were pregnant and 78 (1.31%) were lactating mothers. With regard to WHO staging, 90.33%, 3.84%, 2.56% and 3.27% of the patients were in stage I, II, III and IV, respectively. Even though 94.26% of the patients had good adherence, about 4.05% and 1.69% had fair and poor adherence, respectively (table 1).

Determinants of immunological recovery
Out of the 9687 patients with severe immunosuppression, only 2746 (28.35%, 95% CI 27.45% to 29.26%) had immunological recovery (≥500 cells/mm 3 ) after receiving ART for at least 6 months. Many variables were associated with immunological recovery in the bivariate analyses, including gender, age, patients enrolled in a military facility, WHO stage II and III, good adherence, suspected clinical ART failure, suspected immunological      (table 3).

DISCUSSION
The aim of this study was to identify the determinants of immunological recovery following HAART in severely immunosuppressed HIV-infected adolescent and adult patients at enrolment in Northern Ethiopia. The study showed only 28.35% (95% CI 27.45% to 29.26%) had immunologically recovered (≥500 cells/mm 3 ) after receiving ART for at least 6 months. The adjusted analysis found that gender, age, WHO stage III, suspected treatment failure (clinical, immunological and virological), viral non-suppression, 1e (TDF-3TC-EFV) regimen, 2f (AZT-3TC-ATV/r) regimen and 2h (TDF-3TC-ATV/r) regimen were determinants of immunological recovery among patients with severe immunosuppression at enrolment.
Out of the 9687 patients with severe immunosuppression, 2746 (28.35%, 95% CI 27.45% to 29.26%) had immunological recovery after receiving ART for at least 6 months. This finding is lower compared with a study conducted in Bahir Dar, Ethiopia where 30.8% of patients had CD4 count ≥500 cells/mm 3 . 32 Similarly, other studies have reported that 37.6%-59% of patients recovered immunologically. 33 34 Various research findings have also shown that baseline CD4 cell count <200/mm 3 when starting ART is a risk factor for poor immunological response. [35][36][37][38] This might be due to poor thymic function following ART initiation in severely immunosuppressed patients.
This study revealed that male gender was strongly associated with immune recovery, where male patients were 0.50 times less likely to achieve immune recovery than female patients. There are other studies that indicated female patients have better immune recovery than male patients. 16 20 39-43 However, another research from Sub-Saharan countries indicated that gender was not associated with an increase in CD4 counts. 44 The reported gender difference in CD4 cell count might be due to a sex hormone effect. 45 This difference may be justified as male patients are poor with regard to health-seeking behaviour, leading to lower rates of HIV testing and acceptance of linkage to HIV care after a positive result. 46 In this study, the age categories 20-34, 35-49, and 50 years and above were strongly associated with immune recovery, with these patients 0.33, 0.30 and 0.26 times less likely to achieve immune recovery, respectively, compared with the 15-19 years age category. There is also evidence showing that commencing ART at a younger age may be associated with an improved immunological response. 38 47 48 The Euro-SIDA study also confirmed the inverse relationship between age and maximum CD4 cell response. Initiation of ART at a younger age may favour CD4 cell restoration due to a preserved thymic function. However, there are also other studies which suggest age has no significant association with increase in CD4 cell count. 21 The variation among these studies might be due to differences in study design and time to follow-up for HAART.
This study revealed that WHO stage III was strongly associated with immune recovery, where patients at WHO stage III were 0.68 times less likely to achieve immune recovery than patients at WHO stage I. This evidence was also supported by a similar observation that patients in advanced HIV stage had poor immune recovery as compared with those in the early stages of the disease. 48 A similar study from Tanzania reported that patients at WHO stage III and IV were more likely to achieve an inadequate immune recovery on receipt of ART as compared with those at WHO stage I and II. 49 50 This study revealed that viral non-suppression was strongly associated with immune recovery, where virally nonsuppressed patients were 0.38 times less likely to achieve immune recovery than virally suppressed patients. There was strong evidence in Ghana of having CD4 count <350 cells/ mm 3 after 6 months on ART and having plasma VL >1000 copies/mL. 51 Other studies from Ethiopia, Nepal, Thailand and India reported that patients with HIV RNA level ≥1000 copies/mL were more likely to experience immunological failure at ART follow-up as compared with those who had HIV RNA level <1000 copies/mL. 31 52-54 Another study has also shown that maintaining virological suppression results in a greater increase in CD4 cell count in the long term. 55 This study has also supported that patients with suspected clinical ART failure, suspected immunological ART failure and suspected ART failure on initial VL were 0.13, 0.15 and 0.43 times less likely to achieve immunological recovery, respectively as compared to the test reason of initial VL. These findings agree with the knowledge that viral suppression leads to immune recovery. This is due to the fact that as viraemia increases, the depletion of CD4 cells increases.
In summary, this study showed lower immunological recovery among immunosuppressed patients following HAART for at least 6 months. Being male, age older than  Table 3 Continued VL test (suspected clinical ART failure, suspected immunological ART failure and suspected ART failure on initial VL), and ART drug regimens 1e, 2f and 2h predicted immunological failure. This study was specific to Tigray Region, North Ethiopia; however, the findings can be representative of Ethiopia and other LMICs.
The strength of the study is that it was done using routinely collected public health programme data which can reflect the outcome of the current ART management in severely immunosuppressed patients at enrolment. Additionally, this study was done on a relatively higher sample size, which can be generalised to regional and national levels using appropriate statistical analysis to present the findings. The study however is not without limitations. First, there were no data available when baseline and recent CD4 T cell count was conducted and hence the time when the immunological recovery had occurred is not known. Second, due to the nature of the secondary data, the analysis misses some important variables, such as the existence of coinfection, drug side effects during follow-up, body mass index and the grade of ART experience among HIV-infected patients.

CONCLUSIONS
Immunological recovery following HAART was low among severely immunosuppressed patients at enrolment to care. Gender, age, WHO stage III and viral suppression status were determinants of immunological recovery. Men and adolescents with severe immunosuppression should be identified as groups at higher risk for immunological failure. Therefore, a greater support and intensive counselling should be prioritised to adolescents, men and virally non-suppressed individuals in the Ethiopian healthcare system. Contributors AAD conceptualised and designed the study, and was involved in data analyses, acquisition of data, tabulating the data, interpretation of data, preparing the tables and figures, critically revising the manuscript, final review and drafting the initial manuscript. KMK, YWB, KFA, AGW, AAB, NMB, ABW and TWW were coauthors and contributed to the conception of the study, analyses, and revising and approval of the final manuscript. AAD had the primary responsibility for the final content and was involved in the final review. All authors read and approved the final manuscript.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Map disclaimer The depiction of boundaries on the map(s) in this article do not imply the expression of any opinion whatsoever on the part of BMJ (or any member of its group) concerning the legal status of any country, territory, jurisdiction or area or of its authorities. The map(s) are provided without any warranty of any kind, either express or implied.
Competing interests None declared.
Patient consent for publication Obtained.
Ethics approval Ethical clearance and approval were obtained from the Tigray Health Research Institute (THRI) Institutional Review Board (IRB)/Ethical Review Committee (reference number THRI/00132/19). Permission to use the data was obtained from the Tigray Regional Health Bureau and THRI. The data were from a secondary database on HIV-infected patients for VL monitoring while on combination of antiretroviral treatment. All the baseline and recent CD4 counts were extracted from the database retrospectively, which were entered from the sample referral form. The data were not accessible by any other third party other than the study team. The data carry MRN personal identifiers. Informed consent was waived from the ethics committee.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data may be obtained from a third party and are not publicly available. The data that support the findings of this study are available from the Tigray Regional Health Bureau and THRI but restrictions apply to the availability of these data, which were used under licence for the current study and so are not publicly available. Data are however available from the authors upon reasonable request and with permission from the Tigray Regional Health Bureau and Institutional Review Board (IRB) of Tigray Health Research Institute via institutional. review. board. thri@ gmail. com.
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