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African Journal of Biomedical Research
Ibadan Biomedical Communications Group
ISSN: 1119-5096
Vol. 11, Num. 1, 2008, pp. 33-37
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TOXICOSIS OF NON-STEROIDAL ANTI-INFLAMMATORY AGENTS IN RATS
African Journal of Biomedical Research, Vol. 11, No. 1, Jan, 2008, pp. 33-37
Full Length Research Article
Prevalence of Antibodies
to HAART Agents among HIV Patients in Benin city, Nigeria
Omoregie, R1,2*,
Egbeobauwaye, A2., Ogefere, H1., Omokaro, E.U2 & Ekeh , C.C2.
Departments
of 1Medical Microbiology & Haematology/ Blood Transfusion, School
of Medical Laboratory Science, University
of Benin Teaching Hospital, P.M.B.
1111, Benin City, Edo State, Nigeria.
*Address
for Correspondence
Received: February
2007
Accepted
(Revised): September
2007
Published: January
2008
Code Number: md08005
ABSTRACT
Against
the background that some human immunodeficiency virus (HIV)-infected patients
on the highly active antiretroviral therapy (HAART) still experience anaemia,
some possible reasons for this were investigated. Blood was collected from 50
newly diagnosed treatment naive HIV-infected patients, 100 HIV-infected
patients on HAART and 30 apparently healthy HIV seronegative individuals that
served as controls. Haemotocrit values, red blood cell distribution width (RDW)
and the presence of antibodies to the drugs in the HAART regimen were
determined. The mean ± standard deviation of haematocrit values of HAART naive
HIV patients (36.90 ± 5.61%) and those on HAART (37.20 ± 6.20%) were
significantly lower compared to controls (41.50 ± 5.88%) (p = 0.001), though
the difference between HIV patients on HAART and those that were HAART naive
was not significant (p = 0.836). Although, the RDW of HIV patients were lower
than controls, the difference was only significant (p = 0.026) between controls
(17.00 ± 6.01%) and HAART naive HIV patients (15.00 ± 1.87). A total of 81
(81%) out of the 100 HIV patients on HAART had antibodies to one or more of the
HAART drugs. Antibodies to nevirapine (58%) was higher compared to stavudine
(44.6) and zidovudine (42%) ( p = < 0.05). There was no significant
improvement in haematocrit of HIV patients on HAART over HAART naive HIV
patients. Use of zidovudine, nutritional deficiency and presence of antibodies
to the HAART drugs may have been responsible
Key words: antiretroviral therapy, haematocrit, human
immunodeficiency virus
INTRODUCTION
Highly
active antiretroviral therapy (HAART) therapy entails treatment with a
combination of two nucleoside reverse transcriptase inhibitors and a potent
protease or non-nucleoside reverse transcriptase inhibitors, and has generally
be taken as the gold standard for management of HIV patients (Odunukwe et al.,
2005). However, serious interactions with metabolism of sugar and lipids lead
to the appearance of well documented disorders such as insulin resistance,
abnormalities in lipid metabolism and lipodystrophy, particularly associated
with protease inhibitors (Andrade and Cotter, 2006). This leads to the decline
in the use of protease inhibitors in the HAART regimen. HAART have been reported
to improve haematocrit values of human immunodeficiency virus (HIV) infected
patients, despite documented evidence that these antiretroviral drugs used as
monotherapy have cytopenic effect (Odunukwe et al., 2005). HAART have
also been reported to significantly reduce the morbidity and mortality of HIV
infection (Gea-Banacloche and Lane, 1999). Nevertheless, in the early 2000s
adverse reactions began to appear and started to challenge the goals of HAART
(Andrade and Cotter, 2006). Mildvan (2003) reported that many HIV positive
patients receiving HAART still develop mild to moderate anaemia. The use of
zidovudine, alone or in the HAART combination has been reported to be
associated with anaemia (Moyle, 2002). Inhibition of haemoglobin synthesis and
globin gene transcription have reported as the mechanism by which zidovudine
cause anaemia (Wood et al., 1992), and this could be responsible for
this observation.
Anaemia
is also known to be caused by drugs, through various mechanisms. One of which
is production of antibodies against the drug or its metabolite, The mechanism
involves the drug or its metabolite attaching to the surface of red blood cells
and this triggers antibody production, The antibody produced binds to the drug
on the red cell membrane and the complex activates complement with resultant
cell lysis, or the sensitized cells do not activate complement, but are
destroyed extravascularly by the reticulo-endothelial system(Pertz, 1986) The
purpose of this study was 1) to determine the haemotocrit and red cell
distribution width (RDW) of HIV patient (on HAART and not on HAART) compared
with healthy controls, and 2) to determine the presence of antibodies to the
drugs in the HAART regimen.
MATERIALS AND
METHODS
Study
Population
The study was approved by the Ethical Committee of the
University of Benin Teaching Hospital. Ten milliliters of blood was collected
each from 50 newly diagnosed treatment naïve HIV positive patients (17 males
and 33 females), 100 HIVInfected patients on HAART for 3- 6months (39 males
and 61 females), and 30 age and gender matched apparently healthy HIV
seronegative individuals (13males and 17 females) that served as controls. The
HIV patients were attending HIV clinics in the University of Teaching Hospital
(UBTH) Benin City, Nigeria. Verbal informed consent was obtained from every
subject used in this study. Five milliliters of blood was dispensed in ethylene
diamine tetracetic acid (EDTA) containers and the remaining into plain
containers. The serum obtained from clotted samples in the plain container were
frozen at -20° until needed. The HAART regimen for HIV patients on HAART
consist of zidovudine, stavudine and nevirapine.
Determination
of Haematocit and RDW
Haematocrit
values and RDW of all samples were determined using an autoanalyser Sysmex KX
21 (Sysmex Corporation, Kobe, Japan).
Detection
of Antibodies to HAART Drugs
A
modification of the method described by Petz (1986) was used. Briefly, one
tablet each of zidovudine (Retrovir®, Glaxosmithkline, Spain), stavudine
(Zerit®, Bristol-Myers Squibb, France) and nevirapine (Viramune® Boehringer
Ingelheim, Germany) was dissolved in 10ml of normal saline in 3 separate test
tubes (all drugs were freely donated by Pharmacist Ekiuwa Eribo, Pharmacy
department, UBTH). The drug suspension was centrifuged and the supernatant was
used in the test. Equal volume of 3% washed group O rhesus D negative red cells
and the supernatant of zidovudine solution were placed in a test tube and
incubated at 37oC for 30mins. The drug red cell mixture was washed
4 times with normal saline to remove excess drug. Equal volume of study
subjects sera and drug red cell mixture were placed inside a clean test tube
and incubated at 37o for 1 hour. The mixture was centrifuged after
incubation and agglutination or haemolysis was watched out for. If negative,
the mixture was washed 4 times and antihuman globulin (Plasmatec Laboratories
Limited, United Kingdom) was added. This was followed by centrifugation and
agglutination or haemolysis watched out for.
Controls
were performed in the same way with 1) subject serum and group O red cells
without drugs, and 2) drug red cell complex without subjects serum (both gave
negative results).
The
above procedure was repeated using stavudine and nevirapine separately.
Statistical
Analysis
The
parametric data obtained were analyzed using student t-test. The non
parametric data were analyzed using Chi square(X2) test.
RESULTS
The
haematrocrit of HIV patients were significantly lower than controls (p=0.001),
but did not differ significantly (p=0.836) between HIV patient on HAART (with
mean ± standard deviation of 37.20 ± 6.20%) and does not on HAART (mean ± SD of
36.90 ± 5.61%) (Tables 1 and 2). Although, the RDW of HIV patients were lower
than controls, the difference was only significant (p=0.026) between controls
(mean = 17.00%) and HAART naïve HIV patients (mean = 15.00%).
A
total of 81(81%) out of the 100 HIV patients on HAART had antibodies in their
serum to one or more of the HAART agents. The prevalence of antibodies to
nevirapine (58%) was significantly (X2=6.09, P<0.05) higher
compared with stavudine (44%) and zidovudine (42%) (Table 3).
Presence
of antibodies to more than one of the HAART agents was significantly higher (X2=5.63,
P<0.025) than to one of the agent alone (43% Vs 38% respectively) (Table 4).
HIV
patients not on HAART and controls did not show the presence of antibodies to
HAART drugs in their sera.
DISCUSSION
Anaemia is one of the haematologic complications seen in HIV-infected patients
(Moyle, 2002). HAART has been reported to improved haematocrit values of HIV-
infected patients (Odunukwe et al., 2005). Results from this study
showed a significantly higher haematocrit of control compared with HIV patient
- both on HAART and HAART naïve (p = 0.001) (Table 1 and 2). The cause of
anaemia in HIV patients ars multufactorial. From this study, three possible
reasons were examined.
Table
1: Haematocrit
Values and RDW Of HIV-Infected Patients And Controls.
PARAMETER
|
HIV PATIENTS |
CONTROLS(n =30)
|
HAART
Naïve (n = 50) |
On
HAART (n = 50) |
Range |
X ± SD (95% CI) |
Range |
X ± SD (95% CI) |
Range |
X ± SD (95% CI) |
PCV
(%) |
24.40-48.90 |
36.90 ± 5.61 (35.27,38.63) |
19.60-50.90 |
37.20 ± 6.20 (35.98, 38.35) |
30.00-54.80 |
41.50 ± 5.58 (39.32,43.68 |
RDW (%) |
12.60-20.10 |
15.00 ± 1.87 (14.21, 15.71) |
12.10-35.00 |
15.80 ± 3.02 (15.25, 16.31) |
11.10-36.80 |
17.00 ± 6.01 (15.61, 18.43) |
RDW
= Red Cell distribution width, HAART = Highly active antiretroviral Therapy; ± S.D =
Mean ± Standard deviation, CI = Confidence interval.
Table
2: Statistical
Comparison of Haemotocrit Values and RDW of HIV Patients and Controls
Parameters |
On HAART
Vs
HAART Naive
|
On HAART
Vs
Controls
|
HAART Naive
Vs
Controls
|
PCV
(%) |
P
= 0.836 |
P
= 0.001 |
P
= 0.001 |
RDW
(%) |
P
= 0.081 |
P
= 0.129 |
P
= 0.026 |
Table
3: Prevalence
of Antibodies to HAART Agents Among HIV Patients On HAART (N = 100)
HAART Agents |
Presence of antibodies |
Absence of antibodies |
Zidovudine |
42
(42%) |
58
(58%) |
Stavudine |
44
(44%) |
56
(56%) |
Nevirapine |
58
(58%) |
42
(42%) |
n
= number of HIV patients on HAART
X2
= 6.09, p < 0.05
Anaemia associated with HIV infection has been attributed to bone marrow
suppression (Moyle, 2002; Odunukwe et al., 2005). This may explain the
lower haematocrit to HIV-infected patients. Haematocrit of HIV-infected
patients on HAART did not differ significantly from those that were HAART
naïve (p=0.836) (Table 1 and 2). This finding is not in agreement with those of
Odunukwe et al. (2005) and Belperio and Rhew (2004). The difference
between our result and that of Odunukwe et al. (2005), could be due to
the use of zidovudine as one of the HAART agents by the HIV patients on HAART
in this study. Lamivudine replaced zidovudine in Odunukwe et al. (2005) study.
This is important as the use of zidovudine alone or in combination has been
reported to be associated with anaemia (Moyle, 2002). Inhibition of haemoglobin
synthesis and globin gene transcription and toxicity to bone marrow cells,
particularly, erythroid lines have been suggested as possible mechanisms of
anaemia by zidovudine (Cretton et al., 1991; Weidner et al.,
1992). Thus, the suggestion of Moyle (2002) that HIV-infected patients of
African origin (a risk factor for anaemia in the HIV era) should avoid
zidovudine, may be valid.
Nutritional
deficiencies have been reported to be among the factors that play a role in the
develpoment of anaemia among HIV patients(Volberding, 2000; Odunukwe et al.,
2005) Traditional red blood indices, such as MCV, are used to assess
nutritional deficiency. However, increase MCV in HIV-treated patients often
reflects the use of zidovudine(Moyle, 2002) and not necessarily nutritional
deficiency. This limits the use of MCV to assess nutritional deficiency as the
patients in this study took zidovudine as part of their HAART drugs.The RDW has
been recommended as a biomarker to assess nutritional status. (Romero et al.,
1999). HIV-infected patients irrespective of whether they were on HAART or
not, have lower mean RDW compared with controls (Table 1), although this
difference is only significant between HAART naive HIV-infected patients and
controls ( p = 0.026) (Table 2). Perhaps, nutritional deficiencies - a possible
cause of anaemia (Volberding, 2000), exist in our HIV patients and may
contribute to the lower haemotocrit observed. Wood et al. (2002)
reported inadequate nitrient in-take among a large proportion of their HIV
positve patients. This may also explain the low RDW among HIV patients in this
study. There is evidence that nutritional intervention assists in maintainig
and optimizing nutritional status and immune function, prevents the development
of nutritional deficiency, loss of weight and lean body mass, promotes response
to medical treatment and increases longevity in HIV-infected persons(Oguntibeju
et al., 2006). Also, nutritional supplementation has been shown to
decrease viral load(Bouic et al., 2001; Oguntibeju et al., 2006).
Thus, the HIV patients in this study may benefit from nutritional supplements.
Anaemia
can also result from red cell destruction as a result of antibodies produced
against drugs or their metabolites. A total of 81 (81%) out of the 100 HIV
patients on HAART had antibodies to one or more of the HAART drugs with the
prevalence of antibodies to nevirapine being significantly higher compared to
the other HAART drugs ( p < 0.05) (Table 3). It was also observed that
antibodies to more than one of the HAART agents among the HIV patients on HAART
was significant ( p < 0.025) (Table 4). The mechanism by which the antibody
produced against the drugs causes anaemia involves the drug or its metabolite
attaching to the surface of red blood cells and this triggers antibody
production, The antibody produced binds to the drug on the red cell membrane
and the complex activates complement with resultant cell lysis, or the
sensitized cells do not activate complement, but are destroyed extravascularly
by the reticulo-endothelial system(Pertz, 1986) It is possible that the
non-significant difference in haematocrit between HIV patients that are HAART
naive and on HAART (Table 1 and 2) may be due to the presence of these
antibodies. However, this will require further investigation.
The severity of HIV infection also influences anaemia. However CD4 count and
viral load, which are used to measure HIV progression and severity of
infection, were not done because they were not the primary focus of this study.
Further studies are needed to determine their relationship with presence of
antibodies to HAART drugs.
In
summary, in this study, we did not observed any difference in haematocrit
between HIV-infected patients on HAART and those that were HAART niave. Use of
zidovudine nutritional deficiency and presence of antibodies to the HAART drugs
were observed as possible contributing factors. However, the observation of
presence of antibodies to the HAART agents, as contributing to anaemia in HIV
patients, will require further investigation.
ACKNOWLEDGEMENT
We
acknowledge with thanks the Management of University of Benin Teaching Hospital
for permission to carry out this study. We also acknowledge and appreciate
Pharmacist Ekiuwa Eribo for the donation of the antiretroviral agents used.
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