5 Conclusions
5.2 Native Circulation and AVF
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CHAPTER 5
62
significant. There was no difference in the mean white cell count between the study groups (p=0.1). Although the creatinine values for the three arms were within the reference interval, the values for the HIV infected groups were
63
Table 5.2: Haematological and biochemical parameters of the study participants.
Control mean(SD)
HAART exposed mean(SD)
F test P value
Hb(g/dl) WBC(×109/L) Platelet(×109/L) ALT(IU/L)
Creatinine(mmol/L)
13.97(0.9) 5.16(0.85) 290(62) 11.36(2.14) 62.41(14.38)
11.96(1.86) 5.71(1.47) 263(71.48) 20.04(8.72) 77.19(19.93)
10.75(2.24) 5.80(1.76) 258(88.77) 30.74(15.1) 83.07(23.48)
68.20 4.716 4.064 73.00 23.524
0.00 0.10 0.02 0.00 0.000
Table 5.3: Tukey post hoc analysis of Haemoglobin in study participants.
(I) sarm (J) sarm
Mean
Difference (I-J) Std. Error Sig.
Tukey HSD control on ARV 2.008* .279 .000
ARV naive 3.221* .279 .000
on ARV Control -2.008* .279 .000
ARV naive 1.213* .279 .000
ARV naive Control -3.221* .279 .000
on ARV -1.213* .279 .000
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Table 5.4: Tukey post hoc analysis of WBC in study participants.
(I) sarm (J) sarm
Mean
Difference (I-J) Std. Error Sig.
Tukey HSD control on ARV -.548* .224 .040
ARV naive -.635* .224 .014
on ARV Control .548* .224 .040
ARV naive -.087 .224 .919
ARV naive Control .635* .224 .014
on ARV .087 .224 .919
WBC values were significantly lower in the control group than in the ARV
Table 5.5: Tukey post hoc analysis for platelet count in study participants.
(I) sarm (J) sarm
Mean
Difference (I-J) Std. Error Sig.
Tukey HSD control on ARV 26.914 11.911 .064
ARV naive 31.439* 11.911 .024
on ARV Control -26.914 11.911 .064
ARV naive 4.525 11.873 .923
ARV naive Control -31.439* 11.911 .024
on ARV -4.525 11.873 .923
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Table 5.6: Tukey post hoc analysis for alanine transferase levels in study participants.
(I) sarm (J) sarm
Mean Difference
(I-J)
Std.
Error Sig.
Tukey HSD control on ARV -8.66250* 1.60541 .000
ARV naive -19.36250* 1.60541 .000
on ARV Control 8.66250* 1.60541 .000
ARV naive -10.70000* 1.60541 .000 ARV naive Control 19.36250* 1.60541 .000
on ARV 10.70000* 1.60541 .000
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Table 5.7: Tukey post hoc analysis for creatinine levels in study participants.
(I) sarm (J) sarm
Mean
Difference (I-J) Std. Error Sig.
Tukey HSD control on ARV -14.77500* 3.10392 .000 ARV naive -20.66250* 3.10392 .000 on ARV Control 14.77500* 3.10392 .000 ARV naive -5.88750 3.10392 .142 ARV naive Control 20.66250* 3.10392 .000
on ARV 5.88750 3.10392 .142
Table 5.8 shows the mean and standard deviation of CD4+ lymphocyte cell count and viral RNA copies per ml in the HIV infected participants. There was also a significant difference between both groups (p=0.000).
67
Table 5.8: Mean and standard deviation of CD4+ cell count and viral load of HIV infected participants.
HAART
EXPERIENCED
HAART NAIVE
F TEST SIG LEVEL 1ST Line 2ND Line
Mean CD4+ Cell count/µl
549 (±189)
551(±259) 287(±192) F=35.11 p=0.000
Mean viral load (copies/ml)
197(±16) 177(±59) 8442(±104387) F=25.89 p=0.000 Median viral
load (copies/ml)
200 200 8442(±104387) F=25.89 p=0.000
Baseline clotting profile of study participants
Table 5.9 presents the mean and standard deviation of the clotting profile of participants per group. There was no statistical difference between groups.
Table 5.9: Mean and standard deviation of clotting profiles in study groups
Variable Control mean(SD)
HAART exposed(SD)
F test Sig
PT(secs) 12.46(1.1) 12.57(0.96) 12.55(1.17) 0.29 P=0.764 APTT(secs) 36.39(2.46) 36.31(2.6) 35.94(2.61) 0.724 P=0.486
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Antithrombin, D-dimer, fibrinopeptideA, fibrinopeptide B and PAI-1 levels in study participants.
Table 5.10 shows the mean and standard deviation of antithrombin, D-dimer, fibrinopeptide A, fibrinopeptide B and PAI-1 levels in all study participants. There was a statistical significant difference noted between each group. (p<0.05)
Table 5.10: Mean and standard deviation of Antithrombin, D-dimer, fibrinopeptideA, fibrinopeptide B and PAI-1 levels in study participants.
Variable Control
(SD)
HIV infected ARV exposed (SD)
F test P value
Antithrombin(µg/ml) 133.0(65.) 111.6(49.) 119(48.55) 3.077 0.048 D-dimer(µg/ml) 0.24(0.15) 0.41(0.23) 1.05(0.77) 65.343 0.000 Fibrinopeptide
A(ng/ml)
0.95(0.34) 1.17(0.55) 1.41(0.52) 14.321 0.000 Fibrinopeptide
B(ng/ml)
0.48(0.24) 0.62(0.34) 0.71(0.28) 14.321 0.000
PAI-1(ng/ml) 26.15(7.0) 27.29(6.3) 32.41(6.2) 20.554 0.000
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Comparison of antithrombin levels among study participants.
Table 5.11: Tukey post-hoc analysis for antithrombin in study participants.
(I)study arm
(J)Study arm
Mean
Difference(I-J) Std. Error Sig.
Tukey Control on ARV 21.368475 8.716857 .040
on ARV Control -21.368475 8.716857 .040
on ARV 7.815013 8.716857 .643
Comparison of D-dimer levels among study participants
70
Table 5.12: D-dimer: Tukey Post-hoc analysis in study participants
(I)study arm (J)study arm
Mean
Difference(I-J) Std. Error Sig.
Tukey Control on ARV -.172840 .074506 .055
on ARV Control .172840 .074506 .055
ARV naive Control .808700 .074506 .000
on ARV .635860 .074506 .000
Comparison of fibrinopeptide A levels among study participants
Table 5.13: Fibrinopeptide A: Tukey Post-hoc analysis of study participants
Study arm(I) Study arm(J)
Mean
Difference(I-J) Std. Error Sig.
Control on ARV -.221700 .075984 .011
on ARV Control .221700 .075984 .011
on ARV .245350 .075984 .004
71
Comparison of fibrinopeptide B level among study participants
(I) study arm (J) study arm
Mean Difference
(I-J) Std. Error Sig.
Control on ARV -.145788 .045102 .004
on ARV Control .145788 .045102 .004
on ARV .093712 .045102 .097
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Comparison of plasminogen activator inhibitor-1 levels among study arms
Figure 5.1: Gaussian curve for plasminogen activator inhibitor-1 in HIV infected participants
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significant difference between the HAART exposed group and the control group (p=0.517) (Table 5.15)
Table 5.15: Post-hoc analysis for plasminogen activator inhibitor-1 in study participants.
(I) study arm (J) study arm
Mean Difference
(I-J) Std. Error Sig.
Control on ARV -1.141285 1.039858 .517
on ARV Control 1.141285 1.039858 .517
on ARV 5.117959 1.039858 .000
Doppler ultrasound scan for HIV infected participants
All of the 160 HIV infected persons were requested to have a Doppler ultrasound scan of both lower limbs but only 72 presented as at the time of completion of the study. None had evidence of deep venous thrombosis in both lower limbs.
Prevalence of antithrombin deficiency
The normal reference range for antithrombin was derived from results of the
control participants. This was determined using empirical 95% confidence interval derived from 2.5 and 97.5 percentile confidence limits. Thus participants with values of antithrombin less than 2.5 percentile (56.4µg/ml) were taken to have a
74
deficiency of antithrombin. Figure 5.1 shows the normal distribution (gaussian curve) of antithrombin levels in the control population
Figure 5.2: Gaussian curve for antithrombin
Table 5.16 illustrates the frequency of antithrombin deficiency in the study groups.
In the control group, 1 of 80(1.27%) had antithrombin less than 56µg/ml. 12 out of 80(15%) had antithrombin deficiency in the HAART exposed while 7 out of
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80(8.75%) were deficient of antithrombin in the HAART naive group. Thus out of the 160 participants infected with HIV 20(12%) had antithrombin deficiency compared with 1.27% in the control group. Prevalence of antithrombin deficiency in HIV infected persons is thus 12%. Antithrombin deficiency occurs more
commonly in the HIV infected population (12%) compared with the HIV negative population (1.27%) χ2= 14.35; p= 0.002.
Table 5.16: Prevalence of antithrombin deficiency
Study arm control
on ARV
Count Count Count Total Χ sig ATIII<56.4
ng/ml
ATIII
<56.4
1 12 7 20 14.35 P=0.002
ATIII
>56.4
79 68 71 220
Total 80 80 80 240
Association between PAI-1 with D-dimer
Using Spearman correlation coefficient, PAI-1 level was shown to be positively correlated to the D-dimer level and this was statistically significant. (r=0.226;
p=0.002).
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Association between antithrombin deficiency and D-dimer
Table 5.17: Correlation coefficient of the relationship between D-dimer with PAI-1 and antithrombin deficiency.
PAI-1HIV
ATIIILESS 56.4
Ddimer HIV Spearman's
rho
PAI-1HIV Correlation Coefficient
1.000 -.013 .226
Sig. (1-tailed) . .437 .002
N 160 160 160
ATIII LESS
THAN 56.4
Correlation Coefficient
-.013 1.000 -.253
Sig. (1-tailed) .437 . .001
N 160 240 160
Ddimer Correlation Coefficient
.226 -.253 1.000
Sig. (1-tailed) .002 .001 .
N 160 160 160
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Association between antithrombin deficiency, PAI-1 and fibrinopeptide A and fibrinopeptide B in HIV infected persons.
As illustrated by Table 5.18, there is a negative correlation between antithrombin deficiency and fibrinopeptideA, fibrinopeptide B and plasminogen activator inhibitor-1in HIV infected participants. These correlations were however not statistically significant. There is a positive significant statistical correlation between fibrinopeptides A and B (r=0.184; p=0.010) and between PAI-1 and fibrinopeptide B. (r=0.173; p=0.014)
78
Table 5.18: Correlation coefficient of the relationship between PAI-1 and antithrombin deficiency with fibrinopeptide A and fibrinopeptide B
FPA HIV
FPB HIV
Pai-1HIV AT<56.4 Spearman's
rho
FbpAHIV Correlation Coefficient
1.000 .184 .068 -.065
Sig. . .010 .195 .208
N 160 160 160 160
FbpBHIV Correlation Coefficient
.184 1.000 .173 -.034 Sig. (1-tailed) .010 . .014 .333
N 160 160 160 160
Pai1HIV Correlation Coefficient
.068 .173 1.000 -.013 Sig. (1-tailed) .195 .014 . .437
N 160 160 160 160
ATIII LESS THAN56.4
Correlation Coefficient
-.065 -.034 -.013 1.000 Sig. (1-tailed) .208 .333 .437 .
N 160 160 160 240
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CHAPTER 6
DISCUSSION
Studies have shown that HIV infected persons have a 2 to 10 fold increase in developing venous thromboembolism when compared to the general population.4-6 Predisposing factors include traditional risk factors such as prolonged illness, opportunistic infections and immobilization.
This study excluded participants with such confounders that could independently increase the risk of thrombosis.
Prevalence of Antithrombin deficiency in HIV infected subjects
In this cross-sectional study of coagulation related acute phase proteins in HIV infected participants, the mean antithrombin level was 111.6±49.8ng/ml in
80
The prevalence of antithrombin deficiency in HIV infected participants was 12% in this study. This was a large contrast to the study by Lijifering et al who found a 2%
prevalence of antithrombin deficiency.85 This decrepancy could be due to the fact that this study’s definition of antithrombin deficiency was based on the mean level of 133±65.5ng/ml of the control population. This is lower than the mean
antithrombin level of 150±56.8ng/ml seen in the healthy population as reported by Tait et al.120 This may thus be due to a narrower range of reference value when this is was constructed using ±2SD around the mean.
Also, Lijifering et al measured both antithrombin activity and antigen levels to give more accurate results; the tests were then repeated at a later date to confirm
findings thus improving precision.85 However in this study only antigenic (ELISA) testing was done.
Although cases of antithrombin deficiency in HIV infected patients with
thrombotic events have been reported,87 most cases of antithrombin deficiency are attributed to urinary loss (HIV nephropathy) reduced synthesis (liver disease) amongst others.88 In this study, a history of drug intake and other clinical conditions that may increase risk of thromboembolic event were taken into
consideration and ruled out by the questionnaire administered. Liver function and renal function tests were also carried out to rule out hepatic and renal dysfunction respectively. Although alanine transaminase and creatinine levels were higher in
81
the HIV infected participants than the control population, they were still within reference interval. Thus in this study, there is no evidence to support the fact that the lower antithrombin levels found in the HIV infected participants was due to hepatic or renal disease.
HIV infection induces a low grade persistence inflammatory state and antithrombin being a negative acute phase reactant is expected to be reduced.36 Antithrombin function is also impaired due to a reduced availability of glycosaminoglyans.37 Plasma levels of plasminogen activator inhibitor (PAI-1) in HIV infected subjects
This study found an increase in the level of PAI-1 in the HIV infected participants (27.3±6.36ng/ml) as compared to the control population (26.15ng/ml±6.36). This finding is in keeping with the report of Yki-Jarvinen et al who also found an increase of PAI-1 (28.2ng/ml) in HIV infected participants.121 A further
suggest that HAART can markedly reduce but did not totally normalize the level of positive acute phase reactants such as PAI-I in HIV induced inflammation.
82
This was confirmed by Crum-Cianflone et al who reported that HIV infection is associated with high levels of circulatory inflammatory markers with persisting inflammation long after initiation of HAART.105
Association between PAI-1 levels, antithrombin deficiency and D-dimer levels - a marker of fibrinolysis
In this study there was a positive correlation (r-0.226) between PAI-I and D-dimer levels and this was significant. A similar result was found by Jeremiah et al (r- 0.261, P – 0.002).122
This study also found a negative correlation between antithrombin deficiency and D-dimer levels although this was not significant. The investigator could not assess any study that would support or debunk this finding in HIV infected individuals.
A study by Morse et al also suggested that the raised D-dimer was due to on-going inflammation and endothelial activation which may be mediated by TNF following monocyte/macrophage activation.124
These findings point to the fact that a chronic low grade DIC induced by
inflammation cannot be ruled out as being the cause of the raised plasma levels of D-dimers found in this study. It is important to note however, that lower values of antithrombin in HIV infected patients together with raised PAI-1 levels in the same patients constitute prothrombotic state. Thus one may postulate an ongoing low
83
level thrombosis in these patients and that the thrombi are lysed resulting in raised D-dimer levels documented in this report. That none of the 72 subjects who had Doppler ultrasound scan of the lower limbs showed evidence of thrombosis further suggest that the HIV prothrombotic state (described in this study – low
antithrombin and high PAI-1 levels) are mild and are rapidly lysed.
Association between antithrombin deficiency and PAI-I levels with markers of thrombogenesis - fibrinopeptides A and B.
In this study fibrinopeptide A was noted to be significantly increased in HIV positive participants when compared with control participants. It was also
Also in this study fibrinopeptide B levels followed the same pattern being significantly higher in the HIV infected participants than the control group.
This increase in both fibrinopeptides A and B was also noted in a study by Baker et al.125
Fibrinopeptides A and B are both products of fibrinogen proteolysis during fibrin formation. A raised fibrinogen as well as fibrinopeptide A and B is expected in chronic inflammatory states such as in HIV infection as they are positive acute phase reactants.31
84
In this study PAI-1 and AT levels did not statistically significantly predict
fibrinopeptide A and B levels although there was a correlation. However it can be inferred that fibrinopeptide A and B being products of fibrinogen an acute phase reactant both predict an increased risk of thrombosis. This findings (raised
fibrinopeptide A and B) are in support of the postulated thrombotic occasioned by low antithrombin and raised PAI-1 levels as discussed above.
Presence of asymptomatic/ previously undiagnosed thrombosis in study participants.
Of the 72 patients that presented at the radiology department for Doppler ultrasound scan, none was diagnosed with deep vein thrombosis in both lower limbs. This finding is similar to that done by Sullivan et al.71 Sullivan studied 42,935 HIV infected participants and found an incidence of thrombosis in 1.3 per 1000 in participants with advanced disease while observing over 2.4 years. Saber et al also studied 4752 patients with HIV infection and found DVT in only 0.95%
of them using Doppler ultrasonography. Another study by Laing et al found DVT in 0.96% of 728 participants.126
These studies point to the fact that the incidence of DVT in HIV infection may not be as high as expected.
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CHAPTER 7
CONCLUSIONS
In this study, the HIV infected participants had an increased level of an acute phase reactant PAI-1 and also raised levels of products of thrombin activation of
fibrinogen- fibrinopeptide A and fibrinopeptide B. Antithrombin level was also reduced being a negative acute phase reactant. These measured variables did not satisfactorily predict the presence of thrombosis in these participants. It is now obvious that a complex inter-relationship exists between thrombosis and
inflammation in the setting of HIV infection. A possible mechanism can be a direct triggering of the immune system by HIV and subsequent stimulation of the common pathways involving the inflammatory response and the coagulation system.127
An increased risk of DVT in HIV infected persons is thus probably caused by active ongoing triggering of the immune system by both HIV infection and superimposed occult or obvious infections.
In this study, it was noted that HAART therapy significantly reduced the levels of acute phase reactants but not to baseline levels. Thus HAART therapy can reduce the risk of thrombosis in HIV infected persons.
86
It is obvious that there is an increased risk of thrombosis in HIV infected persons by markers of coagulation (fibrinopeptides A and B) and markers of thrombotic state (low antithrombin and raised PAI-1). Well designed case-control and/ or prospective studies are needed to determine the degree to which HIV infection alters the risk of clotting. If the risk is confirmed the option of treatment may need follow-up with Doppler studies and anticoagulant prophylaxis.
87
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