Analysis of the Coagulation Profile in Children with HIV Infection–Effect of Disease and Anti Retroviral Therapy

Abstract

The pathogenesis of hypercoagulability in HIV infection is multifactorial and usually more than one factor is responsible for a thromboembolic episode. The present study was conducted to evaluate the effect of HIV infection and antiretroviral therapy on various coagulation parameters in paediatric patients. Forty two newly diagnosed paediatric patients with HIV infection who were enrolled at the Anti-Retro viral Therapy (ART) centre of Kalawati Saran Children’s Hospital were included in the study. The patients were grouped into 4 clinical stages according to the WHO clinical staging of HIV disease. Coagulation tests [PT, aPTT, fibrinogen, D-Dimer and coagulation inhibitors i.e. Protein C (PC), Protein S (PS) and antithrombin III (AT III), Lupus anticoagulant (LA) and Anti phospholipid antibody (APLA)] were performed in all the patients at the time of diagnosis and repeated after 6 months. All the patients were started on antiretroviral therapy within 2 months of their diagnosis. At the time of diagnosis, prolonged PT and aPTT were observed in 30.9% and 23% of the cases respectively. Hyperfibinogenemia was seen in 11.9% of patients. D-Dimer was raised in 83.3% of patients. PS, PC & AT activities were reduced in 90.4%, 42.8% & 11.9% of cases respectively. A reduction in the PC and AT activity was seen from clinical stage 1 to 4, but the change was not statistically significant. On follow up after 6 months, a statistically significant reduction in the level of fibrinogen and D-Dimer was seen. Even though there was improvement in the activity of all the coagulation inhibitor after 6 months, statistically significant improvement was seen only for PS. The current study shows that HIV produces a hypercoagulable state in children. Raised d-dimer level and deficiency of natural anticoagulants contribute to the thrombophilic state.

Introduction

Human Immunodeficiency Virus (HIV) infection results from a non transforming retrovirus Human Immunodeficiency Virus (HIV1 &HIV2) which belongs to the family Lentivirus. The virus infects the CD4 + T lymphocytes and impairs cell mediated immunity. Manifestation of the disease ranges from asymptomatic carrier stage to Acquired Immunodeficiency Syndrome which is defined by serious opportunistic infections and malignancies [1].

With the introduction and effective use of Highly Active Antiretroviral Therapy (HAART), the risk of early death from AIDS related opportunistic infections and malignancies have been reduced. Hence, the life expectancy of HIV infected patients is considerably prolonged in the current era [2]. However, newer non-AIDS related complications are emerging as a leading cause of morbidity and mortality in these patients. Arterial and venous thrombosis is being reported at a higher rate and that too in young HIV patients [2]. Studies have reported the frequency of venous thromboembolism (VTE) in HIV infected patients to be 0.19%-7.63% per year, which is 2 to tenfold higher than in an age matched population [3].

The pathogenesis of hypercoagulability in HIV infection is multifactorial and usually more than one factor is responsible for a thromboembolic episode. HIV infection produces a state of chronic immune activation and inflammation. This results in endothelial cell dysfunction, one of the proposed mechanisms of hypercoagulability [1, 2]. Acquired deficiency of natural anticoagulants, i.e. protein S (PS), protein C (PC) and antithrombin (AT) is another important mechanism. Other contributing factors are, raised levels of factor VIII (FVIII), von Willebrand factor (vWF) and presence of antiphospholipid antibody (APLA) [4]. The additional factors for thrombosis in HIV patients are the presence of AIDS associated opportunistic infections and malignancies. The role of antiretroviral therapy on thrombosis is highly controversial. The protease inhibitor group of antiretroviral drugs was found to alter the lipid metabolism thereby increasing the risk of cardiovascular diseases [5–8], however, a few studies found no such correlation between the two [9, 10].

The present study was conducted to evaluate the effect of HIV infection and antiretroviral therapy on various coagulation parameters in paediatric patients.

Materials and Methods

Forty two newly diagnosed paediatric patients (age group of 18 months to 18 years) with HIV infection who were enrolled at the Anti-Retro viral Therapy (ART) centre of a tertiary care hospital were included in the study. The necessary ethical clearance was obtained and informed consents were taken from all the patients. The study period was from December 2013 to April 2015. Patients who were seriously ill / with deranged liver function and/or already on ART were excluded from the study. Patients with past history of thrombosis, any acute thrombotic episode or those on anticoagulant therapy were also excluded. The minimum sample size calculated for this study was 30. The patients were grouped into 4 clinical stages according to the WHO clinical staging of HIV disease [11]. The following tests were performed in all the patients: Complete blood count with peripheral smear, Liver function tests, CD4 count and coagulation tests [PT, aPTT, fibrinogen, D-Dimer and coagulation inhibitors i.e. Protein C (PC), Protein S (PS) and antithrombin III (AT III), Lupus anticoagulant (LA) and Anti phospholipid antibody (APLA)]. All the tests were done at the time of diagnosis and repeated after 6 months. All the patients were started on antiretroviral therapy within 2 months of their diagnosis.

CBC was performed using Sysmex KX- 21 hematology analyser. Liver function tests were done on Beckman Coulter Synchron CX-9 auto analyzer. CD 4 count was done using the BD FACSCALIBUR machine. All clotting assays for PT, aPTT, PC, PS and fibrinogen were performed on fully automated STA compact analyser using STA diagnostic kits. Colourimetric assay of antithrombin III using STA- STACHROM® AT III and Immuno-turbidimetric assay of D-Dimer using STA-LIATEST® D-DI were also done on fully automated STA compact analyser. Detection of Lupus anticoagulant was done using STA®- STACLOT® dRVV Screen and Confirm kits on the same analyser.

Anti phospholipid antibody was measured using Anti Phospholipid Screen ELISA DiaMetra kit. It is an indirect solid phase immunoassay kit for the quantitative measurement of IgG and IgM class autoantibodies directed against β2- glycoprotein mediated anionic phospholipids in human serum or plasma (including Cardiolipin, Phosphotidylserine, Phosphatidylinositol, Phosphatidic acid, Phosphatidyl choline, Lyso- phosphatidyl choline, phosphatidyl ethanolamine).

Statistical Evaluation

Statistical evaluation was done using the Statistical Package for Social Sciences version 17.0. Continuous variables were presented as mean ± standard deviation and categorical variable were presented as absolute numbers and percentage. The comparison of normally distributed continuous variables between groups was performed using student’s t test. Spearman correlation coefficient was used to see the correlation between various variables in cases. P value < 0.05 was considered statistically significant.

Results

At the Time of Diagnosis

The age group of cases ranged from 2 to 17 years with a mean age of 7.64 years and the male to female ratio was 1.62:1. Maximum number of patients (38%) belonged to the clinical stage 3, followed by clinical stage 4 (33%), clinical stage 2 (22%) and clinical stage 1 (7%). CD4 count at the presentation ranged from 71 to 1463 cells/mm³. Out of the 42 patients, eight had CD4 count < 200 cells/mm³ (mean 82.75 ± 53.38 cell/mm³) and rest of the patients had.

CD 4 count > 200 cell/mm³ (mean 550.88 ± 276.72 cell/mm³).

The haemoglobin values at diagnosis ranged from 6.1gm/dl to 13.6gm/dl. Anaemia was present in 80.9% of the patients. The most common type of anemia was microcytic hypochromic followed by dimorphic anemia. Platelet count ranged from 27 to 578 × 10³/µL and 28.5% patients had thrombocytopenia.

No significant difference in haemoglobin value and platelet count were seen between the four clinical stages or patients with CD4 count < 200 or > 200/mm³.

Table 1 shows the results of various coagulation parameters in the patients at the time of diagnosis. The comparison of the coagulation profile between various clinical stages is given in Table 2.

Table 1.

Coagulation parameters of patients at the time of diagnosis

Coagulation parameter	Value at diagnosis	% patients with abnormal values
PT(sec)	14.7 ± 2.56	30.9% (prolonged)
APTT(sec)	37.27 ± 6.94	23% (prolonged)
FBG(mg/dl)	314.93 ± 82.35	11.9% (increased)
D-DIMER(μg/ml)	1.14 ± 0.76	83.3% (increased)
PC(%)	72.43 ± 25.94	42.8% (decreased)
PS(%)	47.05 ± 16.44	90.4% (decreased)
AT(%)	103.88 ± 18.18	11.9% (decreased)

Table 2.

Comparison of the coagulation profile between the four clinical stages

Coagulation parameter	Reference values	Clinical stage 1	Clinical stage 2	Clinical stage 3	Clinical stage 4	p value
PT(sec)	Control ± 2	14.53 ± 5.52	14.7 ± 82	15.3 ± 3.07	14.01 ± 1.29	0.6
aPTT(sec)	Control ± 7 s	37.77 ± 11.98	39.24 ± 7.52	38.19 ± 7.41	34.83 ± 4.63	0.44
D-Dimer (μg/ml)	< 0.5 µg/ml	1.16 ± 0.76	1.3 ± 0.90	0.90 ± 0.68	1.30 ± 0.80	0.46
Fibrinogen (mg/dl)	200–400 mg/dl	240 ± 68.77	342.44 ± 77.64	339.62 ± 74.78	285.07 ± 83.47	0.074
PC(%)	70–130%	100.67 ± 41.86	78.0 ± 22.14	70.0 ± 27.13	65.6 ± 20.96	0.165
PS(%)	70–120%	64.33 ± 22.01	42.78 ± 17.03	41.31 ± 12.52	52.64 ± 16.2	0.051
AT III(%)	80–120%	125 ± 11.36	110.78 ± 15.97	101.69 ± 16.6	97.43 ± 18.99	0. 055

Prolonged PT and aPTT were observed in 30.9% and 23% of the cases respectively. Hyperfibinogenemia was seen in 11.9% of patients. D-Dimer was raised in 83.3% of patients. No difference in above parameters was seen between the clinical stages and with CD4 count.

PS, PC & AT activities were reduced in 90.4%, 42.8% & 11.9% of cases respectively. A reduction in the PC and AT activity was seen from clinical stage 1 to 4, but the change was not statistically significant (Fig. 1).

Fig. 1.

Comparison of protein C, protein S and antithrombin III values between the four clinical stages

Lupus anticoagulant was not detected in any of the patients in the present study. Antiphospholipid IgM antibody was observed in 6.6% of the patients and IgG antibody in 10% of the patients. However, no correlation between PC, PS, AT, LA and APLA was noted.

At 6 Months Follow Up

Thirty out of the 42 patients were followed up 6 months after the diagnosis. All the patients were started on antiretroviral therapy within 2 months of their diagnosis (first line therapy comprising of Nucleoside Reverse Transcriptase Inhibitors and Non Nucleoside reverse transcriptase Inhibitors).

The findings of the follow up group at the time of diagnosis and at 6 months are shown in Table 3.

Table 3.

Comparison of parameters at the time of diagnosis and at 6 months (n = 30)

Test	Day 0	After 6 months	p value
Haemoglobin(gm/dl)	9.54 ± 2.05	11.5 ± 1.37	< 0.0001
Platelet count(X 103 cells/µ l)	283.61 ± 135.24	309.6 ± 104.15	0.4
PT(sec)	14.86 ± 2.65	15.12 ± 6.76	0.84
aPTT(sec)	38.23 ± 6.96	34.35 ± 8.3	0.05
Fibrinogen(mg/dl)	320.40 ± 78.63	278.6 ± 81	0.02
D- Dimer(μg/ml)	1.19 ± 0.79	0.47 ± 0.24	0.001
Protein C(%)	75.40 ± 22.76	85.37 ± 27.8	0.07
Protein S(%)	45.53 ± 14.64	54.9 ± 16.05	0.01
Antithrombin III(%)	104 ± 17.54	110.53 ± 14.92	0.11

A statistically significant reduction in the level of fibrinogen and D-Dimer was seen after 6 months. Even though there was improvement in the activity of all the coagulation inhibitor after 6 months, statistically significant improvement was seen only for PS.

During the follow up period, one patient developed middle cerebral artery thrombosis before starting antiretroviral therapy. Her baseline investigations showed, reduced protein C and protein S activity (46% and 18% respectively), and raised d-dimer level (2.66 µg/ml) and positive antiphospholipid IgG. Unfortunately she was lost to follow up and her sample could not be taken at 6 months.

Discussion

The introduction of HAART was followed by a dramatic change in the spectrum of HIV clinical presentation, with improved survival and emergence of non AIDS complications like thrombosis. Abnormalities of the coagulation system are seen in many patients, deficiency of the natural coagulation inhibitors being the commonest [1, 2, 4].

In the present study prolonged PT and aPTT was observed in 30.9% and 45.23% respectively. However, the mean values were not as raised as the values observed by Omoregie et al. [12] (PT-19.77 ± 2.62 and aPTT- 43.14 ± 5.98 s) and Ifeanyi et al. [13] (aPTT- 42.86 ± 7.10 s).

Raised fibrinogen levels were observed in 11.9% of the study population. Almost similar incidence of increased fibrinogen was reported by Stahl CP et al. (8%) [14].

D-Dimer level was raised in 83.3% of the cases with a mean value of 1.14 ± 0.76 µg/ml and no significant difference was seen among various clinical stages. The levels were very high as compared to the values reported by Pontrelli et al. (0.24 ± 0.17 µg/ml). The latter also observed that levels were higher in patients with higher viral load [15].

Reduced protein C, protein S and antithrombin activity was seen in 42.8%, 90.4% and 11.9% of the patients respectively. The activity of protein C and antithrombin reduced from clinical stage 1 to 4, but the change was not statistically significant. The reported incidence of protein C and protein S deficiencies in adults ranges from 0 to 32% [6, 7, 14, 16–18] and 9% to 76% [8, 14, 19, 20] respectively. Stahl et al. and Erbe et al. did not find any antithrombin deficiency in their study group of adults [14, 17].

Sugerman et al. [18] evaluated coagulation profile in HIV positive pediatric patients before the introduction of antiretroviral therapy. They reported low total PS levels, low antigenic PC and presence of IgG anticardiolipin in 55.9%%, 26.5% and 20.6% of cases respectively. A statistically significant difference in the mean values of total protein S was seen between the asymptomatic group and the AIDS group. All the patients had normal antithrombin level.

Pontrelli et al. [15] evaluated pediatric HIV positive patients after the introduction of antiretroviral therapy. They found protein S deficiency in 51% patients, protein C deficiency in 8% patients and antithrombin deficiency in a single patient (1.1%). Another observation made by them was that children with high HIV viral load had a significantly low activity of all the anticoagulant proteins (protein S, protein C, antithrombin).

At 6 months follow up, a statistically significant reduction in the level of fibrinogen was seen in the present study (p value = 0.02). A recent study by Nasir et al. [21] revealed that ART did not affect the PT and aPTT results and similar findings were noted in the present study also.

Also, a significant reduction in D-dimer level was observed (p < 0.0001). Similar finding was obtained by Wolf K et al. [22] who reported a significant reduction in the D-dimer levels in their patients 5 to 8 months after they were started on antiretroviral therapy.

A statistically significant improvement in anticoagulant activity after the introduction of HAART was seen only for protein S. Although improvement in activity was also seen for protein C and antithrombin it was not statistically significant. Stahl et al. [14] did a study in which they repeated the free and total protein S levels 3 to 6 months after the initial assay and observed no improvement. The fact that their study group was already on HAART during the initial assay could explain the absence of improvement at the second sampling. Erbe et al. [17] performed coagulation tests on newly admitted HIV positive and repeated the tests 10 ± 8 weeks after discharge, and observed a significant improvement in the protein C and protein S activity at the second sampling. They explained that the improvement may be due to antiretroviral therapy and treatment of other opportunistic infections during the hospital stay.

Lupus anticoagulant was not detected in any of the patients in the present study. Many earlier studies have reported high incidence of lupus anticoagulant of up to 72% [23, 24], however most of the recent studies have not found the presence of lupus anticoagulant in their patients [7, 24]. This might be because the earlier authors did not follow the current strict guidelines for the detection of lupus anticoagulant.

Antiphospholipid IgM antibody was observed in 6.6% of the patients and IgG antibody in 10% of the patients in the current study. Similar results were obtained by Erbe et al. [17]. Ivan Palomo et al. [24] also observed anticardiopipin antibody in 12.7% and anti beta-2 glycoprotein in 6.3% of the cases. However, Galrao et al. [23] reported a higher incidence of 44% (IgG, IgM and IgA antiphospholipid antibody). Few past studies reported a correlation between increasing antiphospholipid levels and decreasing protein S and protein C activity [13]. But contrary to this Hassel et al. [20] and Erbe et al. [17] found no such correlation. The present study also did not find any such correlation.

Thus, the current observations like raised d-dimer level, supported that a hypercoagulable state exists in HIV patients. The deficiency of natural anticoagulants contributed to the thrombophilic state. The confounding factors of thrombosis present in adult patients could be excluded in this series as the cases were of paediatric age group. Since patients with deranged LFT were excluded in the study, and the deficiency of the anticoagulant proteins was not uniform (antithrombin deficiency was seen in only 11.9% cases), both these suggests that the cause of reduced natural anticoagulant proteins was not due to the decreased synthesis by liver. Also, since seriously ill patients were excluded from the study and the number of patients with AIDS defining illnesses was very few, the role of opportunistic infections in the hypercoagulable state was minimal. All these factors point to the fact that endothelial dysfunction caused directly by the HIV virus may be responsible for the protein C and protein S deficiency.

The introduction of antiretroviral therapy has changed the natural course of HIV infection to a chronic disease. Acquiring infection during childhood makes the individual exposed to HIV for a very long period with the infection. The current study shows that HIV produces a hypercoagulable state even in children. The direct role of HIV virus along with other confounding factors, (which patients are exposed to during adult life) can cause a very high chance of thrombosis and early death. Also, these children when exposed to stress like major surgeries become more prone for thrombosis. So it is important to keep these children under close monitoring and they can be put under prophylactic anticoagulant therapy when they go through a stressful situation. The improvement of the hypercoagulable state after starting antiretroviral therapy seen in this study, suggests the importance of proper initiation and adherence to antiretroviral therapy. These children should also be made aware of the lifestyle measures they should follow in order to avoid the other contributing factors of thrombosis. Larger studies and long term follow up are needed for further establishing the direct role of HIV in the hypercoagulable state of these patients and to see the role of prophylactic anticoagulant therapy in them.

Author Contributions

Dr Priya Thomas: iterature search, data acquisition, data analysis, manuscript preparation, statistical analysis, Dr Sunita Sharma: Concepts, design, manuscript editing, manuscript review, “Guarantor”, Dr Jagdish Chandra: Definition of intellectual content, clinical studies, Anita Nangia: Data analysis, Shivali Sehgal: Literature search, data analysis, manuscript preparation, manuscript editing.

Funding

The Authors did not receive support from any organization for the submitted work.

Declarations

Conflict of interest

The authors have no conflicts of interest to declare that are relevant to the content of the article.

Ethical Approval

The approval was obtained from the ethical committee of the college. The procedures used in the study adhere to the tenets of the Declaration of Helsinki.

Informed Consent

Informed consent was obtained from the parents/legal guardians.