Immuno-virologic outcomes and immuno-virologic discordance among adults alive and on anti-retroviral therapy at 12 months in Nigeria

The study was reviewed and approved from all the Institutional Review Boards (IRB) of all three index hospitals in Nigeria, the Nigerian National Health Research Ethics Committee (NHREC), and the University of Maryland School of Medicine, Baltimore, Maryland, USA. Written informed consent was obtained from all the study participants after counseling and introduction of the study.

The HIV AIDS Care and Anti-Retroviral Therapy (HACART) study was a large multi-center observational clinic-based cohort study that described the predictors of loss to follow-up, immuno-virologic outcomes, immuno-virologic discordance and sub-optimal drug adherence within the AIDS Care and Treatment in Nigeria (ACTION) project. Funded by the U.S. Centers for Disease Control and Prevention Global AIDS Program (CDC GAP), the ACTION project is a tripartite partnership between the Institute of Human Virology of the University Of Maryland School Of Medicine, the Institute of Human Virology Nigeria, and the Nigerian Federal and State Ministries of Health.

A cohort of 2585 initially ART naïve adults who started HAART between April 2008 and February 2009 were followed up for 12 months in three representative government hospitals in Nigeria: University of Abuja Teaching Hospital, Abuja (UATH), University of Benin Teaching Hospital, Benin (UBTH) and Asokoro District Hospital, Asokoro, Abuja (ADH). These sites consisted of a mix of tertiary (UATH and UBTH) and secondary (ADH) health facilities located in different regions of the country and serving different populations to increase generalizability. With 805(31%) lost to follow-up, a total of 628 out of the 1780 patients alive and active on the program at 12 months were randomly selected for in-depth interviews and laboratory work-up with detailed virologic and immunologic testing (Figure 1). There were no significant demographic and clinical differences between study patients at the 12 month time period and all the other patients alive and active at that 12 month time period (data not shown).

Patients had their information recorded at baseline, were enrolled into ART care and followed up with prospective data abstracted from the medical records. Additional data not available at baseline was obtained at the 12-month visit. Blood work included HIV PCR RNA testing, CD4 cell count test, complete blood count and chemistry. Diagnosis of virologic and immunologic failure was made based on the results of the laboratory testing.

Before commencement of ART most patients underwent detailed history, physical examination, treatment preparation, HIV disclosure counseling, symptomatic screening for tuberculosis and laboratory work-up. Laboratory tests done at baseline included CD4 cell count, complete blood count, liver function tests and serum creatinine test. There were some missing laboratory results due to laboratory reagent shortage, or administrative errors. Cotrimoxazole was provided to eligible patients whose CD4 counts were less than 200 cells/ml. HAART was provided if patients satisfied an immunologic criteria (CD4 less than 200 cells/ml irrespective of WHO staging)^a or a clinical criteria (WHO stage 3 or 4 irrespective of CD4 cell count level). The choice of HAART combination was both guided by national treatment protocols and the discretion of the attending physician but generally consisted of one of three first line regimens (Tenofovir (TDF) + Lamivudine (3TC) + Nevirapine (NVP) / Efavirenz (EFV), Zidovudine (AZT) + 3TC + NVP / EFV, Stavudine (d4T) + 3TC + NVP / EFV. Patients who were anemic were preferably given TDF instead of AZT and persons who were co-infected with tuberculosis were also preferably given EFV instead of NVP. EFV was generally avoided in women of child-bearing age especially if they were not adherent to double contraception methods. A three months’ supply of drugs were provided (after an initial one month supply) with three monthly refill follow-ups and six monthly CD4 testing done together with adherence counseling sessions according to the national ART guidelines. Patients who default from scheduled clinic and pharmacy refill visits were followed-up by the home-based care team.

Standardized patient management and monitoring forms and interviews were used to capture detailed clinical, laboratory and demographic data, some of which were extracted from the CAREWare health information management system. Adherence was measured objectively by pharmacy refill records and subjectively by self-report. Residential distance from treatment sites to closest known town was determined in kilometers using the official Nigerian national road distance calculator and a unique online Nigerian distance tool at Globefeed.com (http://​distancecalculat​or.​globefeed.​com/​Nigeria_​Distance_​Calculator.​asp). Since there were no validated data on socio-economic (SES) classifications in Nigeria, a quintile of class 1–5 was developed based on socio-economic scores calculated using 4 indicators commonly used in social epidemiology studies (highest educational achievement, employment status, household size, and monthly household income in naira (1 US$=150 Nigerian Naira).

Virologic failure (VL^-) was defined as a HIV RNA level of equal or more than 400 copies / ml at the 12 month visit in line with local laboratory assay detection limits (viral load testing was done by quantitative polymerase chain reaction (PCR) for HIV-1 RNA in human plasma using Roche Amplicor version 1.5; Roche Diagnostics, Basal, Switzerland) and national treatment guidelines in Nigeria. Even though baseline HIV RNA levels were not done, it was expected that with adherence to potent HAART regimen, viral load levels would generally be undetectable (< 400 copies/ ml) by 12 months. Fresh samples from UBTH were processed and tested on site and fresh samples from AUTH and ADH were processed and tested at the National IHV reference laboratory onsite at ADH. Immunologic failure (CD4^-) was defined with the WHO definition of “fall in CD4 count to baseline (pre-HAART) or below baseline levels (zero CD4 cell change)” or failure of CD4 cell to improve by at least 50 cells at 12 months [5, 13]. A CD4 change of ≥ 50 cells from baseline was regarded as a sign of progressive immune recovery and has been used in other studies [14, 15]. The CD4 cell count determination was done using Cyflow counter (Cyflow counter, Partec, GmbH, Gorlitz, Germany). Immuno-virologic failure is the combination of immunologic and virologic failure with virologic failure generally seen to precede immunologic failure. In this study, immuno-virologic discordance is said to occur when virologic failure is not followed by immunologic failure or when virologic success is not accompanied by immunologic success. Immuno-virologic discordance was divided into: virologic failure with immunologic success (VL^-/CD4⁺) and immunologic failure with virologic success (VL⁺/CD4^-).

Predictors of interest (age, marital status, residential distance, baseline CD4 cell count, employment, tuberculosis history, regimen type, HIV disclosure, drug adherence and educational achievement) were categorized for data analysis. Hemoglobin was categorized either as a dichotomous variable using hemoglobin cut-off of 10 g/dl for anemia. Kruskal Wallis and Pearson χ² tests were used to compare continuous and categorical variables respectively by site and outcome. Univariate and multivariate logistic regression was used to model the association between potential clinical, demographic, behavioral and socio-economic predictors on virologic and immunologic outcomes. To adjust for confounding, predictors were not included in the multivariable model if they yielded a univariate p-value of ≥ 0.2 according to the methods described by Maldonado and Greenland [16, 17]. Covariates that were a priori known to be associated with the outcome of interest were forced into the model for face validity regardless of level of significance. Covariates in the final model were selected by step-wise backward selection procedure. Model goodness of fit was done by Hosmer-Lemeshow goodness of fit test.

All results were assessed at the 5% level of statistical significance for a 2-sided test. We also compared baseline demographic and laboratory parameters among patients that were sampled at 12 months and those who did not make it to 12 months. Some of the viral load and CD4 samples (6% and 5% respectively) were missing or were lost at random due to laboratory and / or administrative error. All missing data was assumed missing completely at random (MCAR) and multiple imputations (using sequential regression multivariate imputation (SRMI) method) using STATA did not show any differences in study result interpretation. All analyses were done with STATA version 11 (STATA Corp, College Station, Texas).

Figure 1 shows the flow chart and Table 1 summarizes the baseline demographic, clinical, behavioral and socio-economic characteristics of the study participants. The median age at HAART initiation was 35 years (IQR: 30–41 years) and majority of the patients were females (n=400; 63.7%). The baseline median CD4 cell count was 142 cells/mm³ (IQR: 85–223), baseline median hemoglobin level was 10.9 g/dl (IQR: 9.3-12.4) and anemia based on hemoglobin less than 10 g/dl was common (n=192; 35.2%). Majority of the patients were married (n=359; 57.2%). HIV disclosure was high (n=595; 94.7%) and most of the patients (n=402; 64%) lived less than 50 kilometers from their respective treatment sites. More than half of the patients (n=321; 51.1%) were on a TDF-containing regimen at baseline with no substitutions within the 12 months. Even though low educational achievement levels with no formal education were seen in 301 (47.9%) patients, however, socio-economic indicators showed high employment levels (n=524; 83.4%).

There were differences in patients’ characteristics by site on all variables except marital status, HIV disclosure, adherence by self-report, smoking and drug use history. Males differed significantly from females in age at HAART initiation, marital status, TB history, baseline CD4 cell count, baseline hemoglobin count, employment status and smoking and alcohol history. This study was focused on outcomes among those alive at 12 months, and because of the random selection of patients, there were no major differences in the 628 patients selected for the study and the other 1152 who were alive at 12 months and not selected for the study (data not shown). There were differences in the baseline characteristics of age, residential distance from treatment site, baseline CD4 count, baseline anemia status and anti-retroviral drug regimen between the patients who were lost-to-follow-up and those alive and in the study at 12 months (Table 2). The outcomes and associations of patients lost-to-follow-up is in another related paper while this paper focuses on those who were alive at 12 months.

Of the 591 with completed and validated viral load results, 453 had viral load results < 400 copies/ml giving a virologic suppression rate of 76.7% and thus a virologic failure rate of 23.4%. The median baseline CD4 cell count for patients with viral load < 400 copies / ml was 147 cells/mm³ (IQR: 87–225) compared to those with viral load > 400 at 122 cells /mm³ (IQR: 76–196) (p-value 0.04). Of 596 patients with CD4 cell counts (32 samples were not usable due to laboratory mishaps or administrative error) the immunologic failure rate was 22.7%. While 60% (n=338) had ideal immuno-virologic success (VL⁺/CD4⁺), immuno-virologic discordance rate was 33% (Table 2) with 17% (n=94) of the patients having had virologic failure with immunologic success (VL^-/CD4⁺) and 16% (n= 90) of the patients having had immunologic failure with virologic success (VL⁺/CD4^-).

In the univariate logistic model (Table 3), a decreased odds of virologic failure was significantly associated with residential distance 51–100 kilometers (OR 0.45; 95% CI: 0.22-0.92) compared to residential distance less than 50 kilometers, and post-secondary/tertiary educational achievement (OR 0.53; 95% CI: 0.32-0.88) compared with no formal education. An increased odds of virologic failure was significantly associated with anemia with hemoglobin <10 g/dl (OR 1.59; 95% CI: 1.07-2.35) and with poor drug adherence of <95% both by pharmacy refill record (OR 3.76; 95% CI: 2.06-6.87) and self-reported adherence (OR 1.79; 95% CI: 1.07-2.99). After adjusting for site of study and other potential confounders, a decreased odds of virologic failure was still significantly associated with residential distance and post-secondary / tertiary educational achievement (OR 0.60; 95% CI: 0.30-0.86). Also, an increased odds of virologic failure remained significantly associated with age less than 30 years (OR 1.79; 95% CI: 1.17-2.67), anemia with hemoglobin less than 10 g/dl (OR 1.71; 95% CI 1.22-2.62) and adherence by pharmacy refill record < 95% (OR 3.82; 95% CI: 2.17-5.97).

A decreased odds of immunologic failure was not significantly associated with any predictor in the univariate or multivariate regression models (Table 4). However, an increased odds of immunologic failure was significantly associated with age less than 30 years (OR 1.55; 95% CI: 1.01-2.37) in the univariate model, and with male gender (OR 1.46; 95% CI: 1.04-2.45), age less than 30 years (OR 1.50; 95% CI: 1.11-2.39) in the multivariate regression model.

In the multivariate regression model, an increased odds of VL^-/CD4⁺ compared to VL⁺/CD4⁺ was significantly associated with poor adherence as monitored by pharmacy refill records (OR 3.90; 95% CI: 1.92-8.24) and anemia (Hemoglobin < 10 g/dl) (OR 1.80; 95% CI: 1.13-2.85) while decreased odds was associated with post-secondary educational achievement (OR 0.40; 95% CI: 0.22-0.68) (Table 5). Compared with VL⁺/CD4⁺, odds of VL⁺/CD4^- was significantly associated with male gender (OR 1.88; 95 CI: 1.05-3.11) and higher baseline CD4 > 200 (9.90: 95% CI: 2.79-35.10) (Table 6).