GLYCATED HEMOGLOBIN AIc TEST AS SCREENING TOOL TO

ASSESS GLYCEMIC
CONTROL IN HIV INFECTED DIABETIC AND NON DIABETIC
PATIENTS IN ARV CLINICS IN, LAGOS NIGERIA.
BY
DR EYISANMI YETUNDE .C.
DEPARTMENT OF CLINICAL PATHOLOGY
LAGOS UNIVERSITY TEACHING HOSPITAL
IDI-ARABA, LAGOS.
RESEARCH PROPOSAL TOWARDS PARTIAL FULFILMENT OF
REQUIREMENTS
FOR AWARD OF Msc. CLINICAL PATHOLOGY DEGREE.
SUPERVISOR
DR O.O.SORIYAN MBBS (Ibd MWACP (!"b #$d%c%&$ FMCP"'(
(C($#%c"! )"'(*!*+,, !$c'-.$. "&d c*&s-!'"&' c($#%c"! )"'(*!*+%s'
C!%&%c"! )"'(*!*+, d$)' CMUL/LUTH.
l
TABLE OF CONTENTS
CHAPTER ONE
lNTRODUCTlON
CHAPTER TWO
AlMS AND OBJECTlVE
CHAPTER THREE
LlTERATURE REVlEW
CHAPTER FOUR
SUBJECTS AND METHODS
REFERNCES
APPENDlX l: lNFORMED CONSENT FORM
APPENDlX 2: QUESTlONNAlRE
CHAPTER ONE
2
INTRODUCTION
Over 22.9 million people in sub-Saharan Africa are infected with HlV,
representing nearly 70% of the world's total population of people living with
HlV and AlDS (PLWHA).
l
Substantial funding from governments and non-government groups has
led to a dramatic scale-up in provision of highly active antiretroviral
treatment (HAART) across the continent in the last decade.
l

There are now over 5 million Africans on HAART
l
, This is a re-
markable feat given the challenges of providing HlV care in resource-
limited settings. lndeed, HAART has significantly altered the natural history
of this life-threatening condition.
2
As children and adults are living longer on HAART, there is increasing
concern about rising incidence of insulin resistance, glucose intolerance,
type 2 diabetes, and dyslipidemia among PLWHA(People living with aids).
3
Since the advent of HAART, diabetes has become a leading cause of
morbidity for patients with HlV in North America and Europe.
4,5
Emerging
data from across Africa also indicate that the prevalence of diabetes and
dyslipidaemia is increasing as people are living longer on HAART.
6,7
Therefore Keeping track of blood glucose levels is an important part of
health monitoring for HlV positive people. lnternational guidelines
recommend yearly screening of all HlV infected individuals for diabetes
mellitus.
8,9
lt is well known that almost 30% of people with Diabetes mellitus go
undiagnosed
l0
, and nearly 25% of them have microvascular complications
by the time their diabetes mellitus is diagnosed
ll
.Every effort must
3
consequently be made to diagnose diabetes mellitus and the other forms of
glucose intolerance (impaired glucose tolerance (lGT) and impaired fasting
glucose (lFG)) as promptly as possible in PLWHA .
l2
ln 2009 the American Diabetes Association (ADA) introduced glycosylated
hemoglobin Alc (HbAlc) as an alternative to fasting blood glucose (FBG)
and 2-h oral glucose tolerance tests (OGTT) in screening for type 2
diabetes mellitus.
l3,l4
One of the most common markers of chronic glycemia is hemoglobinAlc
(HbA
lc
)
l5
However, little is known in this environment about whether the
use of HbA
lc
results in diagnosis of more cases of glucose abnormalities in
the HlV positive population than the fasting blood glucose test (FBG)
alone.
This study was carried out to evaluate the effectiveness of HbAlC in
assessing glycemic status in HlV patients in a sub Saharan population. The
complex and multifactorial nature of glucose metabolism deregulation
makes management of hyperglycemia or diabetes mellitus challenging in
HlV-infected patients. lt is hoped that a set of recommendations will be
developed in this environment to guide practitioners in assessing, treating,
and monitoring hyperglycemia or diabetes mellitus in HlV-infected patients
from this study.
CHAPTER TWO
4
AIMS AND OB0ECTIVES
AlMS
To determine the performance of glycated hemoglobin as a screening test
for glycemic status and monitoring of glycemic control respectively in a
group of non diabetic and diabetic HlV infected persons at ARV clinics in
Lagos metropolis.
OBJECTlVES
To determine the pattern of glucose abnormalities in HlV infected
patients.
To determine the prevalence of glucose abnormalities in HlV patients.
Determine the sensitivity and specificity of HbAlC as a screening test
compared to fasting blood glucose (FBG) in HlV infected persons.
To determine the association between HAART regimen, CD4 counts,
and PCV on HbAlC assay results.
CHAPTER THREE
5
LITERATURE REVIEW
Since the introduction of highly active antiretroviral therapy (HAART)
in the mid-l990s, the mortality and morbidity of HlV/AlDS has decreased
substantially
l6
. ln fact, HlV/AlDS has become chronic illness for many
patients on HAART, with the increased survival of these patients; there
have emerged a number of unexpected consequences of chronic illness,
especially in the form of metabolic disease.
l6
The viral burden and stress that are present in HlV-infected patients elicit a
complex hormonal and immunologic response that may alter various
biochemical pathways, including glucose metabolism.
l7
Since the advent of highly active antiretroviral therapy (HAART) in the
mid-l990s, abnormalities in glucose homeostasis have been reported with
increasing frequency in persons infected with human immunodeficiency
virus (HlV)
l8
. lnsulin resistance and glucose intolerance were reported to
be uncommon in HlV-l infection prior to the use of potent antiretroviral
regimens.
EPIDEMIOLOGY
With the marked increase worldwide in the incidence and prevalence
of different forms of diabetes, there has been great interest in determining
the frequency and characteristics of disorders of glucose metabolism
(diabetes, impaired glucose tolerance (lGT) etc), in HlV-infected patients.
l9
The epidemiology of DM has been studied to a limited extent in
antiretroviral-nave HlV patients. ln a study which enrolled 4l9 racially
diverse antiretroviral-nave patients, the baseline prevalence of DM was
2.6%].
20
A similar group of patients had a higher baseline prevalence of
diabetes mellitus when co infected with hepatitis C virus (HlV/HCV co
infected group 5.9% vs. 3.3% HlV alone)]
2l
. ln the Women's lnteragency
HlV Study, HlV-infected women reporting no recent HAART showed a DM
incidence rate of l.53/l00 person-years; those reporting HAART containing
a protease inhibitor (Pl) had a DM incidence of 2.50/l00 person-years, and
6
those reporting non-Pl-containing HAART had a DM incidence of 2.89/l00
person-years. The incidence of DM among HlV-uninfected women was
l.96/l00 person-years
22
.
Most epidemiological data regarding DM and lGT come from studies
of patients on HAART
23
. An early study of HlV patients with lipodystrophy
reported a 35% prevalence of lGT and a 7% prevalence of DM, compared
to 5% and 0.5% respectively for matched non-HlV controls
24
. The
Multicenter AlDS Cohort Study showed a l4% incidence of DM in HlV-
infected men exposed to HAART, which was four times higher than among
HlV-seronegative controls
25
. Other groups have reported l3.2% incidence
of insulin resistance (lR) in HlV-infected children
26
, and l3% incidence of lR
after l year of HAART
27
. The prospective D:A:D study, composed primarily
of patients on HAART, showed that the incidence of DM increased with
cumulative exposure to HAART (particularly stavudine, a thymidine
analogue) with a relative risk of l.ll after adjustment for other potential risk
factors
28
. Several groups have studied the prevalence of metabolic
syndrome (for which one possible criterion is impaired fasting glucose or
lGT) in HlV-infected individuals. One group showed that the prevalence of
metabolic syndrome was higher among HlV-infected patients on
antiretroviral therapy (ART) than among non-HlV-infected healthy controls
(l5.8% vs. 3.2%)
29
. A Spanish study estimated the prevalence of metabolic
syndrome to be l7% in HlV-infected patients receiving HAART
30
. ln the
Women's lnteragency HlV Study, metabolic syndrome was more prevalent
in HlV-seropositive than HlV-seronegative women (33% vs. 22%,
respectively)
3l
. Samaras et al, who also studied HlV-infected individuals on
HAART, found the prevalence of metabolic syndrome to be l4% to l8%
(using lnternational Diabetes Federation or the National Cholesterol
Education Program's Adult Treatment Protocol lll criteria, respectively)
32
.
Data from relatively small studies and heterogeneous HlV
populations suggest that metabolic (including glycemic) abnormalities
among HlV patients may be affected by ethnicity
33-36
. HbAlc levels in HlV
patients are associated with older age and ethnicity
37
. To date, no studies
have systematically addressed the effect of ethnicity and its interaction with
glycemic parameters in HlV patients.
7
To address this issue, data collected in the Heart Positive study was
analyzed - a large, multiethnic study of hypertriglyceridemic but otherwise
healthy patients with HlV infection on stable HAART all of whom reported
no history of diabetes and underwent oral glucose tolerance testing
38-39
.
African-Americans had significantly greater impairment of glucose
tolerance, as indicated by higher 2-hour glucose and HbAlc levels, than
either Hispanics or Non-Hispanic Whites (NHWs). After adjusting for age,
sex, HlV duration, HAART duration, smoking, obesity, fasting glucose,
fasting insulin and lipid levels, African-Americans and Hispanics had
significantly higher HbAlc and 2-hour glucose levels than NHWs [Misra,
Balasubramanyam, et al, unpublished data].
These findings are important because there is also an increased risk
of myocardial infarction (Ml) in HlV infected patients, and both metabolic
syndrome and dysglycemia are risk factors for Ml. A higher Ml incidence
rate was seen in HlV-positive men exposed to Pls for l8 months or more
40
.
ln HlV-infected patients on HAART, the incidence of Ml increased with
longer exposure to therapy (adjusted relative rate per year of exposure,
l.26)
4l
An extensive search of hospital databases showed that the rate of
acute Ml was higher in an HlV-infected cohort over non-HlV patients, with a
relative risk of l.75, even after adjusting for age, gender, race,
hypertension, DM, and dyslipidemia
42
.
DISORDERS OF GLUCOSE METABOLISM IN HIV
A spectrum of disorders of glucose metabolism has been associated
with HlV infection and antiretroviral therapy.
lNSULlN RESlSTANCE occurs when the target tissues of insulin action fail
to respond appropriately to insulin, resulting in increased pancreatic insulin
production.
lMPAlRED GLUCOSE TOLERANCE is an elevated blood sugar level of
l40-l99 mg/dL 2 h after receipt of a 75-g loading dose of glucose during an
oral glucose tolerance test.
8
lMPAlRED FASTlNG GLUCOSE occurs when the fasting blood sugar level
is in the l00-l25-mg/dL range.
DlABETES MELLlTUS (DM) is diagnosed when the fasting blood sugar
level is >l26 mg/dL, or the 2-h oral glucose tolerance test glucose level is
>200 mg/dL and is confirmed by additional testing, or when a patient has
symptoms of DM (frequent urination, thirst, blurred vision, or weight loss) in
the setting of a blood glucose level >200 mg/dL.
RIS1 FACTORS
Risk factors for the development of disorders of glucose metabolism in
HlV infected people include
Obesity
ln a large multinational cohort analysis of HlV-infected individuals,
diabetes rates were two-fold higher in obese participants, compared with
those with normal body mass index (BMl).
43
Fat distribution is more
predictive than BMl for incidence of diabetes: patients with HlV and higher
sex-appropriate waist hip ratios are also far more likely to develop
diabetes.
44
Lipodystrophy
lt reflects profound defects in lipid turnover kinetics
45, 46
and clearly
induces severe insulin resistance and the tendency to hyperglycemia.
Hyperinsulinemic-euglycemic clamp studies have demonstrated that
compared to non-lipodystrophic patients, HlV patients with lipodystrophy
have decreased insulin-stimulated glucose disposal
47,48
, increased
intramyocellular lipid , and impaired skeletal muscle glucose
uptake
49
Lipodystrophic patients also have increased fasting plasma levels
of free fatty acids and insulin
50
, and a higher percentage of hepatic fat
(associated with increased serum insulin levels)
5l
.
Older age
9
Older people (>65 years) with HlV are up to four times more likely to
develop diabetes compared with those under the age of 50 years.
52
Race
While there has been a great deal of research into how race affects the
development of diabetes in westernized nations, very little is known about
the complex interplay of race and other societal factors on the development
of diabetes among PLWHA in Africa.
53


Use of most Pls, NRTl exposure (particularly Stavudine)
54
Hepatitis C virus co-infection
HlV co-infection with HCV is associated with a higher prevalence of
hyperglycemia. This correlates with the fact that HlV/HCV co-infected
patients on HAART demonstrate greater insulin resistance, higher levels of
activated platelets, and more endothelial dysfunction than HlV patients
without co-infection
55
. When patients with HlV/HCV co-infection are placed
on Pl-based HAART, they have significantly increased risk of new-onset
hyperglycemia
56.57
vitamin D deficiency
58
concomitant medications e.g. niacin, growth hormone,
corticosteroids,pentamidine and antipsychotics
59
sex hormones
Low serum Testosterone was associated with impaired fasting glucose and
DM, independent of waist circumference and BMl in a large multiethnic
cohort
60
. The association between low Testosterone and DM and insulin
resistance is likely mediated by body fat, specifically by visceral adipose
tissue. Adipose-derived factors inhibit the hypothalamicpituitarygonadal
(HPG) axis, which decreases Testosterone, which promotes further
accumulation of adipose tissue. This in turn promotes further insulin
resistance. Testosterone may also act independently by decreasing muscle
insulin sensitivity
60
.

Hyper androgenic conditions, such as polycystic
l0
ovarian syndrome (PCOS), have been strongly associated with glucose
intolerance and insulin resistance in women
60a
. Sex hormonebinding
globulin (SHBG), a serum protein that affects free circulating hormone
levels, has been inversely associated with insulin resistance and type 2
diabetes in both sexes.
60
CD4 count.
There does not seem to be a direct association between the CD4 count or
the duration of infection and the risk of developing diabetes
6l, 62
. There is
also conflicting evidence as to whether CD4 nadir is associated with an
increased risk of diabetes. ln one recent analysis, HlV-infected men with a
lower nadir CD4 count had an increased risk of incident glucose
abnormalities compared with those with higher nadir CD4 counts
63, 64
.
However, in another French cohort study of HlV-infected patients followed
for l0 years, there was no association between CD4 nadir and the onset of
diabetes.
62

'Return to health' phenomenon.
With the introduction of anti-retroviral, most HlV-infected patients
experience an improvement in their general health. This return to health is
associated with weight gain, improved appetite, and increased caloric
intake. This in turn can lead to insulin resistance and diabetes.
PATHOGENESIS OF GLUCOSE DISORDERS IN HIV
The mechanisms of glycemic deregulation and associated defects in
insulin action and secretion in HlV-infected patients are numerous and
complex, with new information continually surfacing.
ll
lmpairment of glucose metabolism is thought to result predominantly from
tissue insensitivity to the effect of insulin (insulin resistance). A
compensatory increase in insulin secretion is needed to inhibit hepatic
gluconeogenesis and to increase muscle uptake of glucose.
Multiple mechanisms are likely to contribute to insulin resistance in
the HlV-positive patient taking ART. These mechanisms are likely to
involve the direct effects of antiretroviral medications, the indirect
consequences of fat redistribution, chronic inflammatory changes induced
by HlV, and hepatic steatosis.

EFFECTS OF ANTIRETROVIRAL MEDICATIONS
Nucleoside reverse transcriptase inhibitors (NRTls)eg Stavudine,
Zidovudine,
Lamivudine, abacavir, tenofovir, emtricitabine, didanosine strongly linked
to the development of diabetes.
65
ln the 'D: A: D' (data collection on
adverse events of anti-HlV drugs) cohort, exposure to Stavudine was
associated with l9% relative risk of developing diabetes. Exposure to
Zidovudine and didanosine also increased the risk
66
. These drugs pre-
dispose to diabetes development as a consequence of the following
mechanisms:
(a) Mitochondrial toxicity. Several NRTls directly affect mitochondrial
function.
67
ln turn; mitochondrial dysfunction has been implicated in the
pathogenesis of insulin resistance. Short-term exposure to Stavudine, for
example, can reduce insulin sensitivity in healthy volunteers.
68
As
treatment guidelines are being updated the NRTls most commonly
associated with these side effects are being phased out.
69

(b) Lipodystrophy. Several NRTls, including Stavudine and Zidovudine,
are associated with development of lipodystrophy, which itself is
associated with accelerated onset and increased prevalence of
diabetes
70, 7l
. ln a large cohort study, the 'Lipodystrophy Case Definition
Study', the prevalence of diabetes was 7% in those with lipodystrophy
and 3% in those without.
7l
Factors associated with lipodystrophy include
CD4 count at ART initiation and ART duration.
l2
(c) Pancreatitis. Rarely, certain NRTls can cause pancreatitis. This in turn
can lead to the development of diabetes.
Protease inhibitors (PlS) e.g. indinavir, ritonavir, lopinavir saquinavir,
atazanavir, darunavir. This class of drugs were the first HlV medications
to be implicated in the pathogenesis of glucose abnormalities among HlV-
infected patients.
72
Subsequent research has demonstrated that individual
Pls have different capacities to induce insulin resistance and that risk of
diabetes is dose-
73
and duration-
62
dependent. Hyperglycemia resolves in
almost all patients where Pls are discontinued
74
. Evidence suggests that
these drugs exert their effect by a number of mechanisms.
(a) Down regulation of GLUT-4,
75
the transporter responsible for
movement of glucose into fat cells and cardiac and skeletal muscle, has
been identified as one possible mechanism for diabetes in patients on
certain Pls, such as ritonavir.
(b) Inhibition of peroxisoe proliferators-acti!ated receptor g acti!ity,
leading to reduced adipocyte differentiation is another proposed
mechanism
74
.
(c) There is evidence that certain Pls, including saquinavir and ritonavir,
lead to a reduction in beta cell function of between 25% and 50%
76
. The
exact mechanism for this remains unclear.
Non-nucleoside reverse transcriptase inhibitors (NNRTlS). Eg Efavirenz,
nevirapine, etravirine in most African countries, first-line regimens include
one of the NNRTls, either efavirenz or nevirapine. Studies have
demonstrated that this class of drug is occasionally associated with
development of dyslipidaemia.
77
Nevirapine is linked to increases in low-
density lipoprotein (LDL) concentrations
78
whereas long-term use of
efavirenz may lead to increases in total cholesterol and triglycerides
79
.
Research has not demonstrated an association between this class of drug
and diabetes.
EFFECTS OF ABNORMAL FAT REDISTRIBUTION
New data suggests that accumulation of lipid within the
intramyocellular (lMCL) compartment may also contribute to glucose
l3
intolerance. ln this regard, increased lipolysis and deposition of fatty acids
within the myocytes may interfere with insulin signaling through effects on
Pl3-kinase. ln addition, decreased Adiponectin has been demonstrated in
association with reduced extremity fat and increased visceral adiposity in
HlV-infected patients
80
. Adiponectin increases the oxidation of fat within
the muscle. Reduced levels of Adiponectin are associated with decreased
insulin sensitivity and this mechanism may also contribute to altered
glucose regulation in HlV-infected patients.
CHRONIC INFLAMMATORY CHANGES INDUCED BY HIV
During any infectious process, the release of cytokines may affect
glucose metabolism. ln HlV infection, there is an increased release of TNF-
o, lL-6 and lL-8 by both infected T cells and adipose tissues these
cytokines can induce inflammatory insulin resistance and are associated
with increased levels of HOMA-lR
8l
. Activities of the HlV-l accessory
proteins Vpr and Tat have potential roles in initiating or aggravating insulin
resistance. Vpr has been shown to inhibit PPAR-y-mediated transcriptional
co-activation in !itro (which might induce an inflammatory or lipotoxic form
of insulin resistance in adipose tissues and liver)
82
and to decrease specific
transcriptional effects of insulin such as inhibition of phosphoenolpyruvate
carboxykinase (PEPCK) expression
83
, which would promote
gluconeogenesis and fasting hyperglycemia. Tat is known to activate
NFkB, which would induce TNF-o production, block FFA uptake by
adipocytes and suppress insulin receptor signaling all of which could
potentially decrease glucose disposal by blunting Glut4 translocation, and
promote insulin resistance by increasing serine phosphorylation of insulin
receptor substrate-l (lRS-l)
84, 85
. ln HlV patients initiating HAART, levels of TNF-o after 48 weeks of
treatment were associated with incident DM
85
.
G!,c"'$d H$#*+!*b%& A2c "s Sc.$$&%&+ 3*. D%"b$'$s M$!!%'-s %& HIV-
I&3$c'$d I&d%4%d-"!s.
Regular screening for diabetes is essential for all patients with HlV,
especially those who are on HAART
86
. ln 2009 the American Diabetes
l4
Association (ADA) introduced glycated hemoglobin AlC (HbAlc) as an
alternative to fasting blood glucose (FBG) and 2-h oral glucose tolerance
tests (OGTT) in screening for diabetes. They defined diabetes as having an
HbAlc 6.5%
87, 88
. As with FBG and OGTT, this cut-off was based on
increasing risk of retinopathy
89
. Measuring HbAlc has several advantages
over glucose measurements.
Glycated hemoglobin (heoglobin "#c$ %b"
#c
$ "#&, or %b
#c
;
sometimes also HbA2c) is a form of hemoglobin that is measured primarily
to identify the average plasma glucose concentration over prolonged
periods of time approximately previous 8-l2weeks
90, 9l
. lt is formed in a
non-enzymatic glycation pathway by hemoglobin's exposure to plasma
glucose
92
. Normal levels of glucose produce a normal amount of glycated
hemoglobin. As the average amount of plasma glucose increases, the
fraction of glycated hemoglobin increases in a predictable way. This serves
as a marker for average blood glucose levels over the previous months
prior to the measurement. HbAlc is a specific subtype of HbA. Glucose
binds slowly to Hemoglobin (Hb) and produces glycosylated Hemoglobin.
There are several types of glycosylated hemoglobin measures (including
total glycosylated Hb and HbAl), but HbAlc, which is formed when HbA is
glycosylated, is now considered the best and standard measure.
Hemoglobin AlC was first separated from other forms of hemoglobin
by Huisman and Meyering in l958 using a chromatographic column. lts
increase in diabetes was first described in l969 by Samuel Rah bar et al'
The 20l0 American Diabetes Association Standards of Medical Care in
Diabetes added the Alc > 48 mmol/mol (>6.5%) as another criterion for the
diagnosis of diabetes
93
.
Advantages in the use of HbAlc for screening and diagnosis of diabetes-
.
l .Patient does not need to be fasting.
2. HbAlc concentration is related to the development of complications
94, 95.
3. HbAlc has a smaller intra-individual biological variability (within 2%)
respect to that of plasma glucose (l2-l5%)
96, 97
and l6.6% for OGTT
98
.
l5
4. HbAlc is not influenced by sudden glycemic variations (such as under
stress) and reflects the mean plasma glucose levels over the last 2-3
months.
5. HbAlc suffers a limited influence from common drugs known to influence
glucose metabolism.
6. HbAlc is already used as an important target for therapy and is familiar
to clinicians.
The majority of manufacturers of HbAlc kits have already
standardized the assay kits to the current reference systems.
99, l00
Clinical trials, such as the Diabetes Control and Complications Trial
(DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS)
have shown that improving HbAlc measures will decrease the
development and progression of eye, kidney and nerve complications in
both type l and type 2 diabetes mellitus.
The use of HbAlc has its limitations, ADA guidelines suggest HbAlc is an
acceptable screening test unless there are abnormalities in erythrocyte
structure (e.g., hemoglobinopathies)
l0l
or turnover (e.g., pregnancy
l02
,
significant bleeding, hemolysis, or iron deficiency anemia)
l03
.
Using HbAlc to rule out diabetes in subjects at risk of developing the
disease could significantly reduce the number of other tests (e.g., OGTT)
performed and result in a better diagnostic sensitivity and specificity.
A number of techniques are used to measure HbAlC.Laboratories use:
High-performance liquid chromatography (HPLC): The HbAlc result
is calculated as a ratio to total hemoglobin by using a chromatogram.
lmmunoassay
Enzymatic
Capillary electrophoresis
l6
Point of care (e.g., doctor's office) devices use:
lmmunoassay
Boronate affinity chromatography
ln the United States, AlC testing laboratories are certified by the
National Glycohemoglobin Standardization Program (NGSP) to standardize
them against the results of the l993 Diabetes Control and Complications
Trial (DCCT).
Higher levels of HbA
lc
are found in people with persistently elevated blood
sugar, as in diabetes mellitus.
The approximate mapping between HbA
lc
values given in DCCT
percentage (%) and eAG (estimated average glucose) measurements is
given by the following equation
eAG(mg/dl) = 28.7 AlC 46.7
eAG(mmol/l) = l.59 AlC 2.59
Data in parentheses are 95% confidence intervals
Tab l
HbA
2c
$AG ($s'%#"'$d "4$."+$
+!-c*s$
(5

(##*!/#*!

(##*!/L (#+/dL
5 3l 5.4 (4.26.7) 97 (76l20)
6 42 7.0 (5.58.5)
l26 (l00
l52)
7 53 8.6 (6.8l0.3)
l54 (l23
l85)
8 64 l0.2 (8.ll2.l)
l83 (l47
2l7)
9 75 ll.8 (9.4l3.9)
2l2 (l70
249)
l0 86
l3.4 (l0.7
l5.7)
240 (l93
282)
l7
ll 97
l4.9 (l2.0
l7.5)
269 (2l7
3l4)
l2 l08
l6.5 (l3.3
l9.3)
298 (240
347)
l3 ll9 l8.l (l52l)
326 (260
380)
l4 l30 l9.7 (l623)
355 (290
4l0)
l5 l40 2l.3 (l725)
384 (3l0
440)
l6 l5l 22.9 (l926)
4l3 (330
480)
l7 l62 24.5 (2028)
44l (460
5l0)
l8 l73 26.l (2l30)
470 (380
540)
l9 l84 27.7 (2332)
499 (4l0
570)
l8
CHAPTER FOUR
SUB0ECTS AND METHODS
STUDY DESIGN
This study will be a prospective study of the sensitivity and specificity
of HbAlc as a screening and monitoring test compared to fasting blood
glucose (FBG), among HlV-infected diabetic and non-diabetic patients.
(antiretroviral-treated and treatment nave.)
STUDY POPULATION
Recruitment for the study will be at ARV clinics in Lagos
metropolis.HlV positive people who fulfill the set criteria will be categorized
as 'patients'.
PATIENT SELECTION
lnclusion criteria
Patients aged l8 and above.
Both male and female patients.
Patients with confirmed HlV seropositivity by western blot.
Patients who are diabetic.
l9
Patients who have commenced antiretroviral therapy for at least
6months will be recruited as 'treated' group.
Patients who have not commenced antiretroviral therapy will be
recruited as 'treatment- nave' group.
Exclusion criteria
Pregnant women and nursing mothers.
Patients with hemoglobinopathy.
Patients with recent changes in antiretroviral therapy.
Patients with recent blood loss or blood transfusion.
Patients with anemia.
Polycythemia.
SAMPLE SI6E DETERMINATION
Sample size determination will be based on the The formula,
l04
&78
9
):/d
9
n=sample size
z=critical value at 95% confidence level, usually set at l.96
p=Prevalence (l6%)
q=l-p
d=precision of l0%
20
lmputing the variables,
n= (l.96)
2
0.l60.84/(0.l)
2
n=5l.6
A sample size of 5l.6 was obtained
ln order to increase validity of the study an extra 30% will be included for
non responders giving a sample size of 67.l(approx 70) for each group to
be studied.
Therefore a total sample size of l40 will be used (70 for HlV positive
antiretroviral treated, 70 for HlV positive patients not on treatment).
SAMPLlNG TECHNlQUE
The sampling technique will be by stratified random sampling.
Stratification will be by whether patients have been treated or not. Patients
will be randomly selected, each balloting 'yes' or 'no' for selection.
ETHlCS
This study will be reviewed and approved by the research and ethics
committee of Lagos university teaching hospital. lnformed consent will be
obtained from each participant (see appendix l).Anonymity confidentiality
of findings will be ensured. Patients will be allowed the right to decline from
the study, or proceed.
DATA COLLECTlON
This would be done using different research tools after adequate
training and testing. They include:
2l
Questionnaires (see appendix ll) will be administered to study participants
to obtain basic demographic data, history of diabetes mellitus, onset,
duration and type of anti-retroviral therapy family history of diabetes
mellitus, etc.
Physical measurements will be taken, which will include weight in kilograms
(kg), height (cm), waist circumference (cm).BMl (body mass index) will be
calculated from these using the formula BMl=weight/(height)
2
result
obtained will be in kg/m
2
.
Biochemical measures of fasting blood glucose, glycated hemoglobin and
packed cell volume will be done.
lmmunological measures of CD4+ will also be done.
SPEClMEN COLLECTlON AND STORAGE.
Patients will be told to fast overnight (last meal should be l2hrs
before morning collection).Antiseptic preparation of antecubital vein will be
effected with methylated spirit swabs. A tourniquet will be applied to gain
access to vein and about l0mls of venous blood will be collected by
venipuncture into three specimen bottles.
2mls of blood into fluoride oxalate bottle for glucose assay,2mls of blood
into EDTA bottle for packed cell volume (PCV),2mls of blood into EDTA
bottle for glycated hemoglobin assay ,remaining 4mls for CD4+ also into
EDTA bottle.
The specimens will be taken to the laboratory for processing, storage,
and analysis. They will be centrifuged at 3000rpm for 5 mins except the
EDTA whole blood samples. The supernatant (plasma/serum) will be
separated and stored for up to 3 days at 2-8
0
C ;up to several weeks at
-20
0
C in a nonself defrosting freezer.Hemolyzed, icteric and lipemic
samples will be excluded.
22
MATERlALS AND METHODS
GLYCATED HEMOGLOBIN ASSAY- this will consist of the quantitative
estimation of the percent concentration of glycated hemoglobin (HbAlc) in
whole blood.
PRlNClPLE OF GLYCATED HEMOGLOBlN ASSAY-glycated
hemoglobin will be measured using the CLOVER Alc system which
consists of a test cartridge and an analyzer. lt is a fully automated boronate
affinity assay (Boronate beading facilitates the separation of the glycated
hemoglobin fraction from the nonglycated fraction).
The ratio of glycated hemoglobin with respect to total hemoglobin in the
blood sample is calculated using the formula
HbAlc%=A (HbAlc/total hemoglobinl00)+B
A and B are the slope and intercept factor to correct the value for DDCT
(diabetes control and complications trial research group) calibration.
PLASMA GLUCOSE ASSAY
A Glucose oxidase method will be used to quantify plasma glucose levels.
PRlNClPLE OF GLUCOSE ASSAY glucose is determined after
enzymatic oxidation in the presence of glucose oxidase .The hydrogen
peroxide formed reacts under catalysis of peroxidase, with phenol and 4-
aminophenazone to form a red violet guinnoneimine dye as melicate.
Glucose + o
2
+ H
2
o glucose Acid + H
2
o
2H
2
o
2
+ 4-aminophenazone + phenol POD +4 H
2
o
Dye formed is proportional to glucose concentration of sample
Absorbance of coloured complex is proportional to the concentration of
glucose in the specimen. lt will be measured at 500nm.
DETERMINATION OF PAC1ED CELL VOLUME
23
PRlNClPLE- PCV or hematocrit is the percentage of the total volume of
whole blood occupied by packed red blood cells, when a known volume of
whole blood is centrifuged at constant speed for a constant period of time.
DETERMINATION OF CD;< CELL COUNT
This will be measured by the bd facscount fully automated system.
DATA ANALYSIS AND MANAGEMENT
All data forms will be collated and arranged serially .Completed data will be
entered into Microsoft excel and SPSS versionl5.0 (Chicago lL, USA)
statistical package for analysis .Descriptive statistics will be computed with
standard methods and presented as means, standard deviation, frequency
tables and charts. Chi square will be used to compare the association
between o categorical variables.
Student t test will be used to compare the mean between two groups with
Gaussian distribution. A p value of<0.05 will be considered to be
statistically significant.

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APPENDIB (2 CINFORMED CONSENT FORM
GLYCATED HEMOGLOBIN AIC TEST AS A SCREENING TOOL TO
ASSESS GLYCEMIC
37
CONTROL IN HIV INFECTED DIABETIC AND NON DIABETIC
PATIENTS
IN ARV CLINICS IN LAGOS, NIGERIA.
l have fully explained this research to
----------------------------------------------------- and have given sufficient
information, including about risks and benefits, to make an informed
decision.
DATE-------------------------------------------------------
SlGNATURE------------------------------
NAME-------------------------------------------------------------------------------------------
-------
l have read the description of the research or have had it translated into
language
l understand. l have also talked it over with the doctor to my satisfaction. l
understand that my participation is voluntary. l know enough about the
purpose, methods, risks and benefits of the research study to judge that l
want to take part in it. l understand that l may freely stop being part of this
study at any time. l have received a copy a copy of this consent form and
additional information sheet to keep for myself.
DATE----------------------------
SlGNATURE----------------------------------------------------
NAME-------------------------------------------------------------------------------------------
----
WlTNESS' SlGNATURE (lf applicable)
---------------------------------------------------------
WlTNESS' NAME (lf applicable)
-----------------------------------------------------------------
This research has been approved by the health research ethics committee
of the Lagos university teaching hospital. The chairperson of this committee
can be contacted at room l07, l
st
floor Administrative block, Lagos
38
university teaching hospital, lshaga road off ltire road,idi araba, Lagos. ln
addition, if you have any question about your participation in this research,
you can contact the principal investigator,Dr Eyisanmi Y at department of
clinical pathology LUTH,08033892299
APPENDIB (II > QUESTIONNAIRE
Please provide an accurate response to the following questions.The
information provided is strictly confidential. Thank you for your cooperation.
D$#*+.")(%c d"'"
Age (yrs) ----------------
Sex (a) Male (b) Female
Tribe (a) Yoruba (b) lbo (c) Hausa (d) others
Occupation (a) unemployed (b) self employed (c) employee (d) others
Marital status (a) single (b) married (c) separated (d) divorced
Level of education ----------------------------------------------------------------
R%sD 3"c'*.s 3*. d$4$!*)#$&' *3 +!-c*s$ d%s*.d$.s %& )"'%$&'s %&3$c'$d
E%'( HIV.
?. Do you have diabetes mellitus?
(a) Yes (b) no
F. lf yes are you currently on any medication? Please mention
------------------------------------------------------------------------------------------------
---
G. Does any of your parents or siblings have diabetes mellitus? (a) Yes
(b) no
39
@. Have you being diagnosed with hepatitis B before? (a) Yes (b) no
2A. Have you being diagnosed with hepatitis C before? (a) Yes (b) no
22. Are you currently taking any of these medications?
(" Corticosteroids
(b Niacin
(c Antipsychotics e.g. olanzapine
29. Are you currently being managed for any of these diseases?
(" lipoatrophy
(b Hepatitis B and or C
(c Dyslipidaemia
2H. Have you being transfused before? (a) Yes (b) no
2;. lf yes when? ---------------------------------------------
HAART -s$
2=. Have you commenced antiretroviral drugs? (a) Yes (b) no
2?. When did you start? __________________________
2F. What drugs are you currently on? (a)Zidovudine
(b)Lamivudine
(c)Stavudine
(d)Nevirapine
(e)Ttruvada
(f)Efavirenz
40
(g)Lopinavir
(h)Abacavir
(i)lndinavir
(j) Lopinavir
(k)Saquinavir
(l) Others ,specify ___________________________
P(,s%c"! $I"#%&"'%*&
2G. Weight ________________kg
2@. Height _________________m
9A. Waist circumference _______cm
92. BMl ____________________kg/m
2
L"b %&4$s'%+"'%*&s (!"b -s$ *&!,
99. Fasting blood glucose _________________mg/dl
9H .HbAlC ____________________________%
9;. Packed cell volume (PCV) ___________________________%
S'.%c'!, c*&3%d$&'%"!
9=. HlV by determine kit (a) positive (b) negative
9?. Confirmed HlV serology by western blot test (a) positive (b) negative
9F. CD4+ T cell count______________________/mm
3


4l
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