The Hemostatic Balance in HIV-Infected Patients with and without Antiretroviral Therapy: Partial Restoration with Antiretroviral Therapy

Abstract

The incidence of arterial and venous thrombosis in HIV-infected patients is increased compared to healthy controls. In this cross-sectional analysis we measured markers of endothelial cell activation, thrombin generation, fibrinolysis and anticoagulation combined with endogenous thrombin potential (ETP) and activated protein C sensitivity ratio (APCsr) as more global markers. We included 160 consecutive HIV-infected patients with a median age of 46 years (range, 27–77), of whom 92% were male, 74% Caucasian, 11% African American, 9% Hispanic, and 6% Asian. Homosexual contact was the main transmission mode. Seventy percent of patients were using combined antiretroviral therapy (cART). In 83% of patients laboratory markers outside the normal range for a non-HIV–infected population were observed. Significant lower levels of von Willebrand factor (vWF; p = 0.03), factor VIII (p < 0.0001), D-dimer (p = 0.01), and ETP (p = 0.01) were observed in HIV-infected patients on cART compared to patients not on cART. Significant lower levels of protein C (p = 0.05) and free protein S (p < 0.0001), and increased APCsr (p < 0.0001) were found in the HIV-infected patients not on cART. A single association was observed between raised levels of fibrinogen and use of a protease inhibitor (p = 0.002). No significant difference was observed in the percentage of patients with laboratory markers outside the normal range between patients using cART-regimens containing abacavir, stavudine, or didanosine and those with other nucleoside reverse transcriptase inhibitors. Although the prevalence of coagulation abnormalities was lower in HIV-infected patients using cART, a considerable proportion of HIV-infected patients on cART show endothelial cell activation and increased APCsr, suggestive of a persistent procoagulant state.

Introduction

I n both treated and untreated HIV-infected patients the incidence of arterial thrombosis (AT) is increased 2-fold to 6-fold compared to a healthy population of the same age, while the risk of venous thromboembolism (VTE) is 2-fold to 10-fold higher.^1

–4 In general, shared risk factors for atherothrombosis are suggested, such as age, obesity, infections, diabetes mellitus, and metabolic syndrome. Hypercoagulability is likely to be the pathogenic mechanism linking venous and arterial disease.⁵

In HIV-infected patients with a confirmed deep venous thrombosis who went through a thrombophilia screening, high factor VIII, antithrombin, protein C and S deficiency, elevated homocysteine, von Willebrand factor (vWF) and fibrinogen, lupus anticoagulant, anticardiolipin antibodies, antiphospholipid antibodies, and activated protein C resistance (APCsr) without factor V Leiden mutation were observed.^2,6 Inflammatory markers, like high-sensitivity C-reactive protein (hsCRP), are the likely initiators of the coagulation cascade. HIV infection is associated with elevated levels of inflammatory markers like hsCRP.⁷ hsCRP levels are a very good predictor of cardiovascular disease.⁸ It was shown that elevated CRP levels are associated with an increase in markers of inflammation and coagulation.⁹ In AT HIV infection, traditional risk factors, and use of combined antiretroviral therapy (cART) appear to be strongly linked to early onset of coronary heart disease.^10,11 Prolonged exposure to cART, especially protease inhibitors (PIs) and certain nucleoside reverse transcriptase inhibitors (NRTIs), such as abacavir (ABC), stavudine (d4T), and didanosine (ddI), contribute to the increased incidence of myocardial infarction, in part by inducing dislipidemia and diabetes mellitus.^11

–16 The metabolic syndrome has a reported prevalence of 17%–27% in HIV-infected patients with a median age of 40–45 years and even reaches 35% in HIV-infected patients aged 50 or older.¹⁷ Cigarette smoking is a common modifiable risk factor.¹⁷

Since AT and VTE are potentially fatal complications of HIV infection, a better insight is needed into the pathogenesis to identify patients at greatest risk for whom antithrombotic prophylaxis may be warranted. Earlier, mostly retrospective, studies with a small number of patients focused on a selection of coagulation and fibrinolytic markers. Intuitively, a more global coagulation test that integrates the information retrieved from separate coagulation tests could potentially improve our understanding of the pathogenesis and improve patient management. A thrombin generation assay could fill this niche. This assay measures thrombin generation in tissue factor triggered platelet-poor plasma, providing an estimation of the potential to form a clot under pathophysiologic conditions. In that sense, it is different from assays that measure activation markers of clotting including a thrombin-antithrombin complex, which gives an estimation of ongoing clotting at a given point in time. Indeed, in HIV-uninfected patients it has been demonstrated that an increased endogenous thrombin potential (ETP) predicted an increased risk of a first episode of VTE.¹⁸ We combined ETP as a global marker with more specific markers of endothelial cell activation, thrombin generation, fibrinolysis, and anticoagulation in a cohort of HIV-infected patients stratified according to their use of cART to improve our understanding of the origin of the increased incidence of vascular complications.

Patients and Methods

Patients

From December 2005 until December 2007, all consecutive HIV-infected patients treated at the HIV outpatient clinic of the Slotervaart Hospital, Amsterdam, The Netherlands, were asked to participate. Patients using anticoagulant therapy were excluded. After obtaining informed consent, data were collected on age, gender, ethnicity, date of HIV diagnosis, HIV transmission mode, medication use, cART use, duration of cART use, medical history of AT, VTE, and cardiovascular risk factors using a standardized written questionnaire and by reviewing medical records. The study was approved by the Institutional Review Board.

Laboratory studies

Lymphocyte subsets (CD3, CD4, and CD8) were analyzed using flow cytometric techniques (Becton Dickinson, Franklin Lakes, NJ). HIV RNA levels were quantified using the COBAS Ampliprep and COBAS TaqMan (Roche Diagnostics, Almere, The Netherlands). The lower limit of detection for HIV RNA was 40 copies per milliliter. Coagulation assays were performed using citrate plasma. For the measurement of vWF, we used an in-house enzyme-linked immunosorbent assay (ELISA) with antibodies from DAKO (Glostrup, Denmark). The plasma concentrations of prothrombin fragment F1 + 2 (F1 + 2) were measured by ELISA (Siemens Healthcare Diagnostics, Marburg, Germany). Plasmin-α₂-antiplasmin (PAP) complexes were determined by ELISA (DRG Instruments GmbH,Marburg, Germany). Protein C was determined using Coamatic protein C activity kit from Chromogenix (Mölndal, Sweden). Total protein S antigen was assayed by ELISA using antibodies from DAKO. Free protein S was measured in the supernatant after precipitating the C4b-binding protein-bound fraction with polyethylene glycol 8000 and measuring the concentration of free protein S in the supernatant. Coagulation assays (prothrombin time [PT], activated partial thromboplastin time [aPTT], fibrinogen and factor VIII [fVIII]) were performed using an automated coagulation analyzer (Behring Coagulation System, Dade Bering Inc., Deerfield, IL) with reagents and protocols from the manufacturer (Siemens Healthcare Diagnostics). D-dimers were measured by ELISA (Asserachrom D-Di, Roche). The ETP was determined with a calibrated automated thrombogram (CAT). The CAT assays the generation of thrombin in clotting plasma using a microtiter plate reading fluorometer (Fluoroskan Ascent, ThermoLab systems, Helsinki, Finland) and Thrombinoscope software (Thrombinoscope BV, Maastricht, The Netherlands) as previously described.¹⁹ Three parameters were derived from the thrombin generation curve: lag time, peak height and ETP (area under the curve). Data for these parameters were normalized toward normal pool plasma, analyzed on each plate. Resistance to activated protein C (APCsr) was determined by testing the effect of APC on the ETP. The sensitivity to APC (Enzyme Research Laboratories, Swansea, UK) of each plasma sample was determined in both the presence and absence of approximately 4 nM APC (f.c.). The APC concentrations used were adjusted to maintain a residual thrombin generation activity of approximately 10% in normal pooled plasma. Normal pooled plasma was run in parallel on each plate. The normalized APCsr was determined by dividing the APCsr of an individual by the APCsr of the pooled plasma. A normalized APCsr > 1.0 reflects an APC resistant phenotype. Reference values are shown in Table 1 and were derived from a Caucasian, non-HIV–infected control population.

Table 1.

Reference Ranges of Laboratory Parameters

Laboratory parameter	Reference range
PT (sec)	10.7–12.9
aPTT (sec)	25.0–38.0
vWF (%)	50–150
fVIII (%)	63–173
Fibrinogen (g/L)	1.9–4.0
F1+2 (pmol/L)	53–271
D-dimer (μg/L)	150–400
Protein C (%)	70–120
Total protein S (%)	58–130
Free protein S (%)	63–137
PAP-complexes (μg/L)	47–563
ETP (nM·min)	1155–2606
ETP peak (nM)	194–503
APCsr	<1.6

vWF, von Willebrand factor; fVIII, factor VIII; PAP, plasmin-α2-antiplasmin; ETP, endogenous thrombin potential; APCsr, activated protein C sensitivity ratio; PT, prothrombin time; aPTT, activated partial thromboplastin time.

Statistical analysis

Continuous variables were expressed as median values and ranges while categorical variables were expressed as counts and percentages. Nonparametric tests were used for statistical analysis, because most parameters did not follow a normal Gaussian distribution. A p value (two-sided) < 0.05 was considered to be significant. The calculations were performed using the Statistical Package for Social Sciences (version 16.0, SPSS Inc., Chicago, IL) software package.

Results

The patient characteristics are shown in Table 2. There were 160 HIV-infected patients with a median age of 46 years (range, 27–77 years), of whom 92% were male, 74% Caucasian, 11% African American, 9% Hispanic, and 6% Asian. Homosexual contact was the main transmission mode. One hundred twelve patients used cART. Twenty-three patients (21%) used an ABC/d4T/ddI-containing regimen. Of the 50 patients using a PI-containing regimen, 24 patients were using lopinavir/ritonavir and 24 patients atazanavir with or without ritonavir. The laboratory results stratified for use of cART are shown in Table 3. For the whole group, a CD4 cell count below 350 × 10⁶ cells per liter was associated with increased vWF levels (p = 0.04). Furthermore, an HIV viral load above 40 copies per milliliter was associated with increased D-dimer (p = 0.002), vWF (p = 0.01), PAP-complexes (p = 0.03) levels, increased APCsr (p < 0.0001), and free protein S deficiency (p < 0.0001). When the laboratory parameters were compared to reference intervals established with a non-HIV–infected reference group, it was found that 133 of 160 (83%) of patients had one of more laboratory parameters outside the reference range. This was most apparent for vWF (41%), fVIII (31%), F1 + 2 (24%), and D-dimer (18%). Also, 48% of HIV-infected patients displayed an increased APCsr. The majority of abnormal laboratory parameters were found in the group that did not receive cART. Specifically, significantly lower levels of vWF (p = 0.03), fVIII (p < 0.0001), D-dimer (p = 0.01) and ETP (p = 0.01) were observed in HIV-infected patients on cART compared to HIV-infected patients not on cART. Furthermore, significantly lower levels of free protein S (p < 0.0001) and an increased APCsr (p < 0.0001) were found in the untreated HIV-infected patients. We compared coagulation parameters in patients on an ABC, d4T, or ddI-containing regimen with patients receiving other NRTI-containing regimens. No significant differences in evaluated laboratory markers were observed. Patients using a PI-containing regimen had significantly higher levels of fibrinogen (p = 0.002) compared to patients on a NNRTI-containing regimen, whereas the other markers were not significantly different (data not shown).

Table 2.

Patient Characteristics Stratified According to Use of Combined Antiretroviral Therapy

Patient characteristics	Total (n = 160)	cART (n = 112)	No cART (n = 48)
Male gender (%)	147 (92)	102 (91)	45 (94)
Age at study entry, median (range)	46 (27–77)	47 (27–77)	42 (27–72)
Age at HIV diagnosis, median (range)	37 (19–71)	37 (20–62)	37 (17–71)
Ethnicity (%)
Caucasian	119 (74)	82 (73)	37 (77)
Hispanic	14 (9)	12 (11)	2 (4)
Asian	10 (6)	5 (5)	5 (10)
African American	17 (11)	13 (12)	4 (8)
HIV transmission mode (%)
Homosexual contact	130 (81)	92 (82)	40 (83)
Heterosexual contact	18 (11)	10 (9)	6 (13)
Intravenous drug abuse	3 (2)	2 (2)	1 (2)
Other/unknown	9 (6)	8 (7)	1 (2)
CD4 cell count (× 10⁶ cells/L)
Median (IQR)	480 (345–630)	490 (370–650)	450 (330–590)
<350 (%)	40 (25)	26 (23)	14 (29)
≥350 (%)	120 (75)	86 (77)	34 (71)
HIV viral load (copies/mL)
Median (IQR)	40 (40–11,478)	40 (-)	30,864 (9388–91,007)
<40 (%)	92 (58)	91 (81)	—
≥40 (%)	68 (43)	21 (19)	48 (100)
Previous arterial thrombosis (%)	4 (3)	2 (2)	2 (4)
Previous venous thrombosis (%)	—	—	—
Smoking (%)	75 (47)	51 (46)	24 (50)
Use of antihypertensive drugs (%)	9 (6)	8 (7)	1 (2)
Use of lipid-lowering drugs (%)	16 (10)	11 (10)	4 (8)
Use of oral antidiabetics (%)	3 (2)	3 (3)	—
cART use (%)		112 (70)
Duration of cART use (months, IQR)		100 (36–124)
Type of cART
NRTI (%)		3 (3)
NRTI + NNRTI (%)		46 (41)
NRTI + PI (%)		50 (45)
NRTI + NNRTI + PI (%)		9 (8)
PI (%)		4 (4)
ABC/d4T/ddI-containing regimen		23 (21)

cART, combined antiretroviral therapy; IQR, interquartile range; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; ABC, abacavir; d4T, stavudine; ddI, didanosine.

Table 3.

Laboratory Parameters Stratified According to use of Combined Antiretroviral Therapy

	Total (n = 160)	cART (n = 112)	No cART (n = 48)	p value ^a
Laboratory parameters^b
vWF (%)	139 (51–368)	127 (51–281)	155 (101–368)
fVIII (%)	137 (68–300)	128 (68–219)	160 (101–256)
Fibrinogen (g/L)	2.9 (1.6–6.6)	2.8 (1.6–6.6)	3.0 (2.1–5.4)
F1 + 2 (pmol/L)	203 (84–1033)	194 (84–1033)	237 (121–538)
D-dimer (μg/L)	232 (79–1255)	194 (79–1208)	327 (157–1255)
Protein C (%)	114 (43–167)	115 (55–167)	107 (43–163)
Total protein S (%)	97 (65–160)	101 (68–155)	88 (65–127)
Free protein S (%)	76 (25–162)	81 (33–162)	65 (25–127)
PAP-complexes (μg/L)	296 (89–1167)	254 (89–1167)	394 (180–948)
ETP (nM.min)	1761 (1076–3148)	1705 (1076–2506)	1878 (1344–3148)
ETP peak (nM)	365 (222–533)	360 (237–483)	370 (239–533)
APCsr	2.63 (0.28–10.62)	2.22 (0.28–10.39)	3.6 (0.57–10.62)
Number of patients with procoagulant changes^c
Increased vWF	65 (41)	39 (35)	26 (54)	0.03
Increased fVIII	49 (31)	23 (21)	26 (54)	<0.0001
Increased fibrinogen	28 (18)	19 (17)	9 (19)	0.86
Increased F1 + 2	39 (24)	23 (21)	16 (33)	0.10
Increased D-dimer	29 (18)	14 (13)	15 (31)	0.01
Protein C deficiency	4 (3)	1 (1)	3 (6)	0.05
Free protein S deficiency	21 (13)	6 (5)	15 (31)	<0.0001
Increased PAP complexes	20 (13)	11 (10)	9 (19)	0.13
Increased ETP	3 (2)	– (–)	3 (6)	0.01
Increased ETP peak	4 (3)	1 (1)	3 (6)	0.06
Increased APCsr	77 (48)	42 (38)	35 (73)	<0.0001

p value indicates difference between patients with and without cART (χ² test).

Laboratory parameters are expressed as median values (range).

Number of patients (%) with laboratory parameters outside the reference range.

cART, combined antiretroviral therapy; vWF, von Willebrand factor; fVIII, factor VIII; PAP, plasmin-α₂-antiplasmin; ETP, endogenous thrombin potential; APCsr, activated protein C sensitivity ratio.

Three patients experienced a documented vascular event during the 2-year inclusion period. One patient had a venous thrombosis of the leg. This patient was not using cART at the time of the event. Laboratory testing preceding the event showed increased vWF and fVIII levels in combination with decreased free PS levels. Two patients suffered from a myocardial infarction. Both patients were using cART containing a PI. Laboratory testing preceding the event revealed no abnormalities in one patient and an increased fibrinogen level in the other.

Discussion

In this study the spectrum of endothelial cell activation, coagulation, anticoagulation, and fibrinolysis was studied in a large cohort of HIV-infected patients. Our results indicated a lower prevalence of coagulation abnormalities in HIV-infected patients using cART compared to those not using cART. The differences in plasma included lower levels of endothelial cell activation, coagulation, and fibrinolytic markers and higher levels of the anticoagulant proteins. However, in up to 50% of patients persistently abnormal levels were observed despite use of cART, a situation that is still consistent with a prothrombotic state, although at a lower level. No consistent evidence was found suggesting a specific prothrombotic effect of different cART regimens. Together, these results suggest that cART use in our study is overall associated with an improved hemostatic balance that outweighs the effects of possible minor procoagulant properties of certain specific drug regimens.

In our study a CD4 cell count below 350 × 10⁶ cells per liter was associated with endothelial cell activation expressed by elevated vWF levels. Feffer et al.²⁰ stratified HIV-infected patients according to CD4 cell count less than 200/mm³, 200–400/mm³, and greater than 400 mm³. They observed a significant difference in coagulation parameters including higher vWF levels in HIV-infected patients with a CD4 cell count less than 200 mm³ compared to those with a CD4 cell count of greater than 400 mm³. A detectable HIV viral load and no use of cART were associated with identical abnormalities in markers of endothelial cell activation, coagulation and anticoagulation. Selected markers were studied previously in patients before and after the start of cART. A significant decrease in vWF and D-dimer levels was observed, but no normalization after the start of cART.^21,22

In HIV-infected patients with a confirmed deep venous thrombosis who underwent thrombophilia screening, high factor VIII, antithrombin, protein C and S deficiency, elevated homocysteine, vWF and fibrinogen, lupus anticoagulants, anticardiolipin antibodies, antiphospholipid antibodies and APCsr without factor V Leiden mutation were observed.^2,6 Together, the abnormalities observed in our study may well explain the increased incidence of venous thrombosis. In large cohort studies prolonged exposure to cART, especially protease inhibitors, was associated with an increased incidence of myocardial infarction. We observed significantly elevated fibrinogen levels in patients on a PI-containing regimen. An association between elevated fibrinogen levels and increased risk of myocardial infarction was described by Wannamethee et al.²³ Current or recent use of the NRTIs ABC and ddI also seemed to be associated with an excess risk of myocardial infarction.^15,16 In addition, common cardiovascular risk factors including male gender, older age, current or former smoking, previous cardiovascular disease, higher serum cholesterol levels and triglyceride level, and the presence of diabetes have been implicated.^10,11 The increased risk of myocardial infarction associated with PI use was partly explained by dislipidemia.¹⁰ A common mechanism underlying atherothrombosis in HIV has not been clarified yet, but vascular inflammation was suggested. In support of the inflammation mechanism, levels of hsCRP, and interleukin (IL)-6 were higher in patients receiving abacavir, while levels of amyloid A and P and the coagulation markers D-dimer and F1 + 2 were not significantly different.¹⁵ Our study also observed no significant difference in the percentage of patients with coagulation markers outside the normal range between patients that were using ABC/d4T/ddI-containing regimens compared to those with other NRTIs.

Our study included both measurements of previously studied markers as well as determination of novel global coagulation tests including thrombin generation measured as ETP or after addition of APC, as APCsr. We observed significantly lower ETP levels in patients using cART while peak ETP levels were not significantly different. However, in absolute values there was not a clear difference between patients on and off cART and the number with test results outside the reference range was actually low. Another marker that could be used as a global coagulation test is APCsr. APCsr is associated with an increased risk of venous thrombosis by a decreased anticoagulant activity of APC.²⁴ Acquired APCsr could be the result of changes in several parameters including free protein S, fVIII, or lupus anticoagulant,²⁵ while congenital APCsr is associated with the factor V Leiden mutation. APCsr was significantly higher in patients not using cART. We assumed that most HIV-infected patients in our study have an acquired APCsr, since the prevalence of congenital APCsr is approximately 5%–8% in most European studies.²⁶ Of the patients with increased APCsr 14% had a combined elevated fVIII levels and free protein S deficiency, 31% had only elevated fVIII levels, 8% had only free protein S deficiency, and in 47% of patients no abnormalities in fVIII or free protein S levels were observed. Lupus anticoagulants were not measured in this study. The reported prevalence of lupus anticoagulant in HIV-infected patients is generally low, but prevalences up to 72% were documented.^27
–29 Majluf-Cruz et al.⁶ observed increased APCsr in 7.1% of HIV-infected patients with a VTE. No other studies on APCsr in HIV-infected patients have been described so far. APCsr and increased risk of vascular complications have been found in patients with systemic lupus erythematosus and primary cytomegalovirus infection.^30
–32

In addition to their possible role in vascular complications, certain coagulation and inflammatory markers are associated with mortality and disease progression. Kuller et al.³³ presented data on biomarkers associated with mortality from the SMART study. At study entry levels of hsCRP, IL-6, and D-dimer were significantly higher in deaths than in matched controls. In a multivariate model IL-6 and D-dimer remained significantly associated with all-cause mortality. Persistent elevation in fibrinogen and fVIII levels and free protein S deficiency were more prevalent in patients with AIDS than in those with non-AIDS–defining illness.³⁴

The precise mechanism of the prothrombotic state is likely multifactorial and based on the interplay between inflammation and coagulation. Elevated vWF and fVIII levels indicate that endothelial cell activation is a prominent finding in our study, as was also demonstrated in other studies.^22,35 Persistence of viral replication as well as smoking are suggested as important factors. Endothelial cell activation promotes platelet activation, inflammatory reactions, and progression of atherosclerosis.³⁶ Upon activation of the endothelial cell TF is expressed through the nuclear factor-κ B (NF-κB) pathway with subsequent activation of the coagulation system. Due to endothelial dysfunction less thrombomodulin is expressed leading to less activation of protein C. Furthermore increased fVIII levels and free protein S deficiency are associated with increased APCsr. The suggested mechanisms giving rise to a decreased free protein S level in HIV-infected patients remains controversial. In previous studies C4b-binding protein levels were not increased.^37,38 A correlation with anticardiolipin antibodies was suggested.^37,38 In clinical studies not related with HIV a reduced level of free protein S has been associated with increased activity of elastase, presumably released from neutrophils.³⁹ It is possible that also in HIV infection increased production of elastase or other proteolytic enzymes influences the concentration of natural anticoagulants including protein S. Another factor in the prothrombotic state, which we did not study, might be platelet activation and aggregation. The platelet activation markers soluble CD40 ligand and p-selectin were significantly higher in HIV-infected subjects than in HIV-negative control subjects and did not decrease after initiation of therapy.²² Finally, arterial and venous thrombosis share identical clinical risk factors such as age, obesity, diabetes and the metabolic syndrome.⁵ The metabolic syndrome is increasingly recognized in HIV-infected patients.^17,40

There are several limitations to our study. The study is an observational study that cannot distinguish between causal and secondary effects. The study was designed to measure markers of endothelial cell activation, coagulation, anticoagulation and fibrinolysis in a group of chronically HIV-infected patients on and off cART in order to improve our understanding of the mechanism of vascular complications as a long term complication of chronic HIV infection. Therefore, this study did not focus on changes in coagulation parameters over time or before and after the start of cART. Also, given the low numbers of vascular complications no conclusions could be drawn about the association of such complications with any of the measured variables.

In conclusion, HIV infection is associated with a prothrombotic state. The prevalence of coagulation abnormalities is lower in HIV-infected patients using cART compared to those not using cART. However, even a considerable proportion of HIV-infected patients on cART show endothelial cell activation and APC resistance, suggestive of a persistent procoagulant state. No consistent evidence was found suggesting a specific prothrombotic effect of different cART regimens. The true association between endothelial cell activation, increased APCsr, and increased incidence of vascular events in HIV-infected patients should be studied in future prospective studies.

Footnotes

Acknowledgments

We would like to thank J.F.P. Wagenaar, M.D., H. Paap, D.J. Vlasblom, M. Stroomer, and L. van Belle, specialist HIV nurses, for their great help in including patients. Furthermore, thanks to O. Ternede and M. de Rijk, trial nurses, for the work-up of the blood samples. E. Jong is medical consultant for Gilead Medical Sciences.

Author Disclosure Statement

No competing financial interests exist.

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