Anemia and iron homeostasis in a cohort of HIV-infected patients in Indonesia

This cohort study was conducted at Hasan Sadikin hospital as the referral hospital for HIV in West Java, Indonesia. As one of the first 25 hospitals selected by the Indonesian government to provide HIV-care, Hasan Sadikin hospital has delivered free antiretroviral treatment and PCP-prophylaxis since December 2004. Following WHO and national guidelines [24, 25], ART is indicated for patients presenting with WHO clinical stage IV, WHO clinical stage III with a CD4 count below 350/mm³, or WHO clinical stage I or II with a CD4 count below 200/mm³. The national program provides a choice of nevirapine (NVP), efavirenz (EFV), zidovudine (ZDV), stavudine (d4T) and lamivudine (3TC) as first-line ART. For this study, we included all HIV-positive patients above 14 years old presenting between September 2007 and August 2009. All patients signed informed consent and the study was approved by the hospital ethical committee.

At time of first presentation at the hospital, all patients (both ART-naïve and ART-experienced) have a 'baseline visit' for structured interviewed and laboratory examination. Afterwards they come for scheduled visits, once-monthly when on ART, three- to six-monthly when not on ART. ART naïve patients were compared with patients with a favorable effect of ART, excluding those that with less than three months of ART, those who had ever interrupted ART for more than one month, those who had detectable HIV-RNA after six months ART, and those taking 2^nd ART. At presentation, data collected included gender, age, history of injecting drug use, body mass index (BMI, kg/m²), WHO stage, oral candidiasis, and history of ART or tuberculosis (TB) treatment. For ART-experienced patients, treatment duration and regimen were recorded. During follow-up, death was recorded from medical records.

Laboratory examinations included hemoglobin, red cell indices (Cell Dyne 3000, Abbot) and manual reticulocyte counts. Reticulocyte index was defined as the reticulocyte percentage × (measured hematocrit/normal hematocrit) [26]. Anti HCV antibodies were detected by an electrochemiluminescence assay, ECLIA (Elecsys 2010, Roche) and CD4-cell count by flowcytometry (BD Biosciences, Jakarta, Indonesia). In our setting, HIV-RNA (real time PCR, Abbott, USA) is only measured in patients taking ART, but not in ART naïve patients. Between January 2008 and June 2008 we also measured plasma ferritin (ECLIA method Elecsys 2010, Roche; reference range 30-400 ng/mL for men and 35-150 ng/mL for women), soluble Transferin Receptor (sTfR) (enzyme Immunoassay method, Diamed, Eurogen; reference range 1870-2450 U/mL) and high sensitive C-Reactive Protein (hsCRP) (immunoturbidimetry method, Hitachi 912, Roche; reference < 5 mg/L) in newly diagnosed patients. We defined ferritin as 'high' if it was above the upper reference value.

WHO/ACTG criteria were used to define mild (Hb 10.5 - 12.99 g/dL for men; and 10.5 - 11.99 g/dL for women), moderate (Hb 8.0 - 10.49 g/dL) and severe (Hb < 8.0 g/dL) anemia [27, 28]. As diagnosis of TB is difficult in HIV-positive patients in this setting due to its paucibacillary nature and the fact that many patients are unable to expectorate sputum [29], we considered all patients receiving TB treatment within three months after presentation as having TB co-infection. Mortality data were derived from clinical files, reports from community organizations or phone interviews from the clinic. Patients not returning for more than three months without confirmation of death or transfer were considered lost to follow-up. Data are presented as proportions or median with inter quartile range if not normally distributed. Categorical and continuous data were compared using the Chi-squared and Kruskal-Wallis tests respectively. Progression to death was measured by Kaplan-Meier estimates with Cox-regression to examine factors in baseline visits that affecting survival. Ferritin and sTfR concentration divided based on manufacturers reference values. Cox-proportional hazard regression model was used to investigate association between iron inflammatory parameters and mortality. Clinical parameters were examined as a possible determinant of mortality and high ferritin concentrations using logistic regression models. Variables which were significantly associated in univariate analysis (p < 0.05) were used in the final backward stepwise multivariate regression model. Proportionality was first assessed by generating time dependent covariates of interactions between predictors and survival function. Log-transformed ferritin and sTfR values were used to examine the relation between ferritin, sTfR, CRP and CD4. All statistical analysis was done using SPSS version 16.0.2 and Prism 4 for Windows.

A total number of 869 HIV positive patients were enrolled in this study, 70.3% (611) of whom were ART naïve, and 258 (29.7%) of whom were on ART (Figure 1). Table 1 shows the baseline patient characteristics.

In this group, mild, moderate and severe anemia was present in 30.4%, 14.1%, and 4.6%, respectively, while hemoglobin levels were missing for 7 patients (1.1%). Clinical characteristics of ART-naive patients, stratified according to anemia category are presented in Table 1. Most ART-naive patients presented with advanced HIV infection or AIDS; 66.7% had a CD4-cell count below 200 cells/mm³, and 62.8% were in WHO stage III/IV. Malnourishment, defined as a body mass index (BMI) below 18.5 kg/m² was found in 38.0% of patients; severe malnourishment (BMI < 16 kg/m²) was found in 12.7% of patients. One fifth (21.3%) of patients were already on TB treatment or started TB treatment within 3 months of presentation, 58.5% were co-infected with HCV, and oral candidiasis was diagnosed in 27.8% of patients.

Anemia in ART-naïve HIV patients was mostly normocytic, normochromic and characterized by a normal or low reticulocyte index suggesting 'anemia of chronic disease' as the main cause. Anemia was associated with a low CD4-cell count (p < 0.001), and TB treatment (p < 0.001) (Table 1). Hepatitis C co-infection prevalence was not different in patients with and without anemia (56.9% vs. 59.6%, p = 0.54). During follow-up, 314 patients (51.4%) started ART after a median of 28 days (IQR: 19-55). The majority of patients (70.1%), none of whom had moderate or severe anemia, were started on a ZDV-containing regimen. From this group, 13.6% developed anemia during follow-up and were subsequently switched from ZDV to d4T.

Anemia was associated with increased mortality during follow-up. When 611 ART-naïve patients were followed for a median of 4.4 (IQR: 0.0-12.1) months (total follow-up: 335 person-years), 45 patients (7.4%, IQR: 5.3%-9.5%) died and 121 (19.8%, IQR: 16.6%-23.0%) were lost to follow-up (Figure 1). Mortality (75.6% of the total) and loss to follow-up (92.6% of the total) were highest in the first 6 months after presentation. Using Kaplan-Meier estimates, the survival rates at 6 months were 98.6% (95% CI: 97.3-99.9%) for patients without anemia, 95.1% (92.0-98.5%) for patients with mild anemia and 82.5% (75.5-89.4%) for patients with moderate or severe anemia (P < 0.001; Figure 2). Beside a CD4 cell count below 50/mm³ (HR: 5.7, IQR: 1.6-17.8, p = 0.003), moderate to severe anemia remained independently associated with an increased risk of death in multivariate analysis (HR: 6.5, IQR: 2.0-21.2, p = 0.002) (Table 2), This association remained significant after eliminating subjects with tuberculosis co-infection, which is an important risk factor for death.

Serum ferritin, sTfR, CRP and reticulocyte index were measured in a subgroup of 141 randomly selected patients. ART-naïve patients (n = 95) with available iron parameters had very high plasma ferritin concentrations (median 641 ng/mL, IQR: 242-1296), moderately elevated CRP concentrations (median 6 mg/mL, IQR: 2-34), low sTfR concentrations (median 1014 U/mL, IQR: 736-1322), and normal reticulocyte counts (median 0.8%, IQR: 0.6-1.5%).

Very high ferritin concentrations were found in subjects with CD4 counts below or equal to 50 cells/mm³ (median 1078 mg/ml, IQR: 550-1824) compared to subjects with CD4 counts above 50 cells/mm³ (median 279 mg/ml, IQR: 41-465, p < 0.001), and in subjects with moderate-severe anemia (median 1223 mg/ml, IQR: 519-2764) compared to subjects without (median 436 mg/ml, IQR: 245-1296. p = 0.07) or with mild anemia (median 532 mg/ml, IQR: 194-1099, p = 0.01). Moderately elevated hsCRP levels were also found in subjects with CD4 counts below or equal to 50 cells/mm³ (median 12.2 mg/L, IQR: 2.0-63.2) compared to subjects with CD4 counts above 50 cells/mm³ (median 4.9 mg/ml, IQR: 1.5-17.2, p = 0.08), and in subjects with moderate-severe anemia (median 18.4 mg/ml, IQR 4.5-94.4) compared to subjects without (median 2.0 mg/ml, IQR: 1.4-5.4, p = 0.001) or with mild anemia (median 6.0 mg/ml, IQR: 1.2-27.8, p = 0.03). Subjects with high ferritin, high hsCRP or low sTfR concentrations showed a higher mortality during follow-up, although this was not statistically significant (Table 3). Low ferritin levels (< 30 mg/ml) were found in 6.3% of patients, all were women with a CD4 cell count above 200 cells/mm³. There was a significant negative correlation between ferritin concentrations and CD4 count (Figure 3).

Predictors of hyperferritinemia in ART-naïve patients were subsequently determined in a univariate and multivariate model (Table 4). CD4 cell count below 50/mm³ and hsCRP > 5 mg/L were associated with hyperferritinemia after correction for gender, degree of anemia, and HCV co-infection. After exclusion of patients with tuberculosis co-infection, low CD4 count was the only factor associated with hyperferritinemia.

At time of enrollment in the cohort, 258 patients were already taking 1^st line ART, for a median of 24.0 months (range: 3.3 - 96.3 months). The majority of patients were on a ZDV containing regimens (62.8%), the others on a d4T containing regimen. Anemia was less common in patients taking ART: 14.3% had mild, 1.9% had moderate, and no patient had severe anemia (p < 0.001) (Table 1). The use of ZDV was associated with anemia: among patients taking ZDV, 20.3% had anemia, compared with 7.4% among those who were not taking ZDV (p = 0.01). During a median 19.8 months (IQR: 9.8-20.0), 2 (0.8%) ART experienced patients died and 26 (10.1%) were lost to follow-up.

Patients taking ART seemed to have lower plasma ferritin concentrations (median 304.1 ng/mL, IQR; 160.7-574.6) compared to ART-naïve patients, although this was not statistically significant (p = 0.12). In addition, they had lower hsCRP values (median 3.0 mg/mL, IQR: 0.9-6.3, p = 0.02), but similarly low sTfR concentrations (median 1378.5 U/mL, IQR: 1104.3-1798.5, p = 0.95) compared to ART-naïve patients. Duration of ART was not associated with plasma concentrations of ferritin or sTfR (data not shown). There was a significant negative correlation between ferritin concentrations and CD4 count in ART-naïve patients, but not in patients taking ART (Figure 3).