INTERFERON-GAMMA, INTERLEUKIN-10 AND ADIPONECTIN

AS DISEASE PROGRESSION MARKERS IN HIV-1 AND

TUBERCULOSIS CO-INFECTED NON-INJECTION DRUG USERS

FROM MOMBASA, KENYA

SARAPHINE NEKESA NEBERE (BSc.)

Reg. No. I56/22674/2012

Department of Biochemistry and Biotechnology

A thesis submitted in partial fulfilment of the requirements for the

award of the Degree of Master of Science (Medical Biochemistry) in the

School of Pure and Applied Sciences of Kenyatta University.

October, 2015
 ii

DECLARATION

This research is my original work and has not been presented for a degree in any other
university.

Saraphine Nekesa Nebere SignatureDate.

Reg. No. I56/22674/2012

SUPERVISORS
This thesis has been submitted with our approval as supervisors

Prof. Joseph N. Ngeranwa

Department of Biochemistry and Biotechnology
Kenyatta University

SignatureDate

Dr. Tom Were

Department of Medical Laboratory Sciences
Masinde Muliro University of Science and Technology

SignatureDate

Dr. Gerald Juma

Department of Biochemistry
University of Nairobi

SignatureDate
 iii

DEDICATION

This thesis is dedicated to my parents, Mr. Christopher Nebere Okumu and Mrs. Agripina

Oyatsi Nebere, my husband Moses Nyongesa and daughter Racheal Kolwe.

 iv

ACKNOWLEDGEMENT

I wish to express my sincere gratitude to my supervisors Prof. Joseph J.N Ngeranwa, Dr.

Tom Were and Dr. Gerald Juma for their invaluable advice and guidance at all the stages

of the thesis. Special thanks to Dr. Tom Were and Valentine Bundambula for their

expertise in designing and executing this study. More importantly, I pass my appreciation

to the study participants for consenting to the study and clinical and laboratory staff and

management of Bomu Hospital for their support during sample collection. I thank the

Kenya National Commission for Science, Technology and Innovation (NCST/5/003/065),

and Partnership for Innovative Medical Education in Kenya (NIH 1R24TW008889)

grants to Dr. Tom Were and Valentine Budambula for funding this study. Lastly, I

appreciate my family for their support and sacrifice during this study.
 v

TABLE OF CONTENTS
DECLARATION............................................................................................................... ii
DEDICATION.................................................................................................................. iii
ACKNOWLEDGEMENT ............................................................................................... iv
LIST OF TABLES ......................................................................................................... viii
LIST OF FIGURES ......................................................................................................... ix
LIST OF ABBREVIATIONS AND ACRONYMS ........................................................ x
ABSTRACT ..................................................................................................................... xii
CHAPTER ONE ............................................................................................................... 1
INTRODUCTION............................................................................................................. 1
1.1Background information .................................................................................................1
1.2 Problem statement and justification ...............................................................................5
1.3 Research questions .........................................................................................................5
1.4 Hypotheses .....................................................................................................................6
1.5 Objectives ......................................................................................................................6
1.5.1 Specific objectives ..................................................................................................... 6
CHAPTER TWO .............................................................................................................. 7
LITERATURE REVIEW ................................................................................................ 7
2.1 Burden of HIV/AIDS, tuberculosis and drug use ..........................................................7
2.2 Pathogenesis of HIV ....................................................................................................10
2.3 Monitoring of HIV/AIDS disease progression ............................................................11
2.3.1 HIV-1 viral loads in HIV-1/AIDS ........................................................................... 12
2.4 Tuberculosis pathogenesis ...........................................................................................13
2.5 Body mass index in HIV and tuberculosis ...................................................................14
2.6 Role of cytokines in HIV and tuberculosis pathogenesis ............................................14
2.6.1 Interferon-gamma in HIV and tuberculosis pathogenesis ....................................... 16
2.6.2 Role of interleukin-10 in HIV and tuberculosis pathogenesis ................................ 18
2.6.3 Role of adiponectin levels in HIV and TB pathogenesis ........................................ 20
2.7 Drug use and cytokine dysregulations .........................................................................22
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CHAPTER THREE ........................................................................................................ 24

MATERIALS AND METHODS ................................................................................... 24
3.1 Study area.....................................................................................................................24
3.2 Study design .................................................................................................................24
3.3 Ethical considerations ..................................................................................................25
3.4 Sample size calculation ................................................................................................25
3.5 Study population ..........................................................................................................26
3.6 Inclusion and exclusion criteria ...................................................................................27
3.7 Demographic data collection .......................................................................................27
3.8 HIV-1 diagnosis ...........................................................................................................27
3.9 Tuberculosis screening and diagnosis ..........................................................................28
3.10 Measurement of body mass index (BMI) ..................................................................29
3.11 Blood collection and processing ................................................................................29
3.11.1 Determination of HIV-1 viral load ........................................................................ 30
3.11.2 CD4+ T cell measurements .................................................................................... 30
3.11.3 Measurement of plasma interferon-gamma ........................................................... 31
3.11.4 Determination of plasma Interleukin-10 levels...................................................... 32
3.11.5 Measurement of plasma adiponectin levels ........................................................... 33
3.12 Data processing and analysis .....................................................................................34
CHAPTER FOUR ........................................................................................................... 36
RESULTS ........................................................................................................................ 36
4.1 Baseline characteristics of the study participants ........................................................36
4.2 Circulating interferon-gamma levels ...........................................................................38
4.3 Plasma interleukin-10 levels ........................................................................................39
4.4 Interferon-gamma to interleukin-10 ratios ...................................................................40
4.5 Plasma adiponectin levels ............................................................................................41
4.6 Correlations of plasma cytokines with HIV-1 disease markers ...................................42
4.6.1 Correlations in HIV-1 infected patients ................................................................... 42
4.6.2 Correlations in HIV-1 negative individuals ............................................................. 47
 vii

CHAPTER FIVE ............................................................................................................ 51

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ............................. 51
5.1 Discussion ....................................................................................................................51
5.2 Conclusions ..................................................................................................................59
5.3 Recommendations ........................................................................................................61
5.4 Suggestions for future research ....................................................................................61
REFERENCES ................................................................................................................ 62
APPENDICES ................................................................................................................. 71
Appendix I: Approval letter ...............................................................................................71
APPENDIX II: PARTICIPANT CONSENT FORM ........................................................72
APPENDIX III: QUESTIONNAIRE FOR DATA COLLECTION .................................73
APPENDIX IV: MAP OF MOMBASA TOWN ...............................................................74
 viii

LIST OF TABLES

Table 4.1 Baseline demographic, clinical and drug use characteristics ...........................37

Table 4.2 Correlations of cytokines with CD4+ T cell counts, viral loadand BMI in

HIV-1 and TB co-infected ART-exposed patients............................................43

Table 4.3 Correlations of cytokines with CD4+ T cell counts, viral loadsand BMI in

HIV-1 mono-infected ART-exposed patients....................................................44

Table 4.4 Correlations of cytokines withCD4+ T cell counts, viral load

and BMI in HIV-1 and TB co-infected ART- naive patients............................45

Table 4.5 Correlations of cytokines with CD4+ T cell counts, viral loads

and BMI in HIV mono-infected ART-naive patients........................................46

Table 4.6 Correlations of cytokineswith CD4+ T cell counts, and BMI in TB mono-

infected patients.................................................................................................48

Table 4.7 Correlations of cytokines with CD4+ T cell counts and BMI in HIV-1

and TB uninfected non-injection drug users.....................................................49

Table 4.8 Correlations of cytokines, CD4+ T cell counts, and BMI in healthy

controls..............................................................................................................50
 ix

LIST OF FIGURES

Figure 1. Plasma IFN- levels..............................................................................38

Figure 2. Plasma IL-10 levels...........................................................................................39

Figure 3. IFN-/IL-10 ratios in study participants............................................................40

Figure 4. Plasma adiponectin levels..................................................................................41

 x

LIST OF ABBREVIATIONS AND ACRONYMS

Acrp30 Adipocyte complement-related protein of 30 kDA

AFB Acid-fast bacilli

AIDS Acquired immune deficiency syndrome

APC Antigen presenting cells

apM1 Adipose most abundant gene transcript-1

ART Anti-retroviral therapy

ARV Anti-retroviral

BMI Body mass index

CDC Centre for Disease Control and Prevention

COPD Chronic obstructive pulmonary disease

CTL Cytotoxic T lymphocyte

ELISA Enzyme-linked immunosorbent assay

GBP28 28 kDA gelatin binding protein

HCV Hepatitis C virus

HDL High density lipoprotein

HIV Human immunodeficiency virus

HRP Horse-radish peroxidase

IDU Injection drug user

IFN Interferon

IL Interleukin

IQR Interquatile range

 xi

IRIS Immune reconstitution inflammatory syndrome

LDL Low density lipoprotein

MHC II Class II major histocompatibility complex

MTB Mycobacterium tuberculosis

NACC National AIDS control council

NK Natural killer cell

PBMC Peripheral blood mono-nuclear cell

PBS Phosphate buffered saline

PPAR Peroxisome proliferator-activated receptor

TB Tuberculosis

TMB Tetramethyl benzinidine

TNF Tumor necrosis factor

UNAIDS United nations programme on HIV and AIDS

WHO World health organization

 xii

ABSTRACT

Both HIV and TB as well as substance use cause profound dysregulation in the
production of inflammatory cytokines such as adiponectin, interleukin-10 (IL-10) and
interferon-gamma (IFN-). Although there are marked immunologic alterations in HIV
and TB co-infected patients, IFN-, IL-10 and adiponectin levels, and their association
with clinical correlates of disease such as CD4 counts, HIV-1 viral loads and BMI has not
been examined in Kenyan HIV and TB co-infected non-injection substance using
patients. Therefore, this cross-sectional study determined circulating IFN-, IL-10 and
adiponectin levels in substance users at Bomu hospital, Mombasa, Kenya. The study
groups included; HIV-1 and TB co-infected antiretroviral treatment (ART)-naive (n=12)
and -exposed (n=9); HIV-1 mono-infected ART-naive (n=21) and ART-exposed (n=6);
TB mono-infected (n=5); and uninfected substance users (n=32); and healthy controls
(n=27). Demographic, anthropometric and clinical measures were recorded at enrolment
of the study participants. Venous blood was collected from each participant followed by
centrifugation to obtain plasma that was stored at -800C until used for cytokine and viral
load measurements. CD4+ T cell counts were enumerated using a FACSCaliburTM flow
cytometer on whole blood while Abbot m200-RT-PCR was used to determine HIV-1
RNA copies. TB was assessed on sputum using acid-fast staining procedure while plasma
cytokine concentrations were measured using a sandwich ELISA. Statistical comparisons
across-groups for continuous variables were performed using non-parametric ANOVA
(Kruskal Wallis) tests followed by post-hoc Dunns corrections for multiple comparisons.
Spearmans rank correlation tests were used to determine the association of IFN-, IL-10
and adiponectin with viral loads, CD4+ T cell counts and body mass index (BMI). IFN-
levels differed across the study groups (P<0.0001) and were higher in the HIV-1/TB co-
infected ART-naive (P<0.001) and -exposed (P<0.001), TB mono-infected (P<0.001)
and uninfected (P<0.001) substance users compared to healthy controls indicating
influence of substance use on IFN- production. IL-10 levels were different across groups
(P=0.035) and were higher in the uninfected substance users (P<0.05) in comparison
with healthy individuals suggesting that drugs stimulate IL-10 release. On the other hand,
adiponectin levels differed significantly across study groups (P<0.036), and were lower
in HIV-1 mono-infected ART-naive patients (P<0.05) relative to HIV and TB co-infected
ART-nave patients, un-infected non-IDUs and healthy controls depicting adverse effect
of HIV on adiponectin production. Spearman rank correlation tests indicated that IFN-
was positively associated with the CD4+ T cell counts (=0.900; P=0.037) in TB mono-
infected signifying that CD4+ T cells produce IFN- during TB infection. Adiponectin
was positively associated with HIV-1 viral load among ART-naive HIV-1 mono-infected
patients (=0.829; P=0.042) suggesting a link between adiponectin and HIV-1 disease
progression. Altogether, these results suggest that substance use promotes increased IFN-
and IL-10 production in HIV and TB co-infected patients that may trigger disease
progression in this population. Therefore, drug use screening should be adopted in HIV-
1/TB-care centres for effective management of the co-infected patients. Further research
should be carried out to ascertain the role of adiponectin in HIV/TB pathogenesis.
 1

CHAPTER ONE

INTRODUCTION

1.1Background information

Human immunodeficiency virus and acquired immune deficiency syndrome (HIV/AIDS)

and tuberculosis (TB) are the main causes of infectious diseases burden in developing

countries. It is estimated that there were over 35 million cases with 1.6 million deaths due

to HIV/AIDS in 2013 (UNAIDS, 2014). Most of the HIV/AIDS related deaths arise from

opportunistic infections including TB, candidiasis, pneumocystis pneumonia,

histoplasmosis, toxoplasmosis, cryptococcosis, and cancers such as Kaposis sarcoma

(CDC, 2013).

Among these opportunistic infections, TB is the leading cause of death and accounts for

over 25% of all AIDS-related deaths worldwide (WHO, 2014). Globally, Sub-Saharan

Africa is the most affected region contributing 78% of the total estimated 1.1 million HIV

positive new TB cases (WHO, 2014). Kenya is among the 10 countries with highest HIV

and TB co-infection burden, globally. It is estimated that almost 38% of all TB cases in

2013 in Kenya were co-infected with HIV (WHO, 2014). The most viable and currently

used management option for HIV/AIDS is the use of antiretroviral therapy (ART). These

drugs have been reported to increase CD4+ T cell counts among HIV-1 patients, thus

halting disease progression. However, in the absence of ART, HIV overwhelms the

patients immune system increasing the risk of TB infection or reactivation of latent TB.

Therefore, the World Health Organization (WHO) has recommended the initiation of
 2

ART in all HIV and TB co-infected patients regardless of their CD4+ T cell counts

(Mbithi et al., 2014). Unfortunately, in Kenya the proportion of co-infected individuals

on ART is less than 80% (Mbithi et al., 2014), hence, there is need to upscale ART-

treatment among co-infected patients in the country.

Drug abuse is an emerging health problem in the society. It is estimated that almost 5.2%

of the world population aged between 15 and 64 years had used an illicit drug in 2012

(UNODC, 2014). Although comprehensive information on drug situation in Africa is

limited, reports indicate that prevalence of cannabis use in Africa is higher than the

global average (UNODC, 2014). Similarly, in Kenya, drug and substance use has become

a major social and health crisis. Recent statistics show an upward trend in the use of

alcohol, tobacco, bhang, khat, opium, cocaine and heroin among the youths in the country

(Ndetei et al., 2009). The increased prevalence of drug abuse has been attributed to rapid

social change, economic factors, and availability of drugs (Ndetei et al., 2009). Major

towns in Kenya such as Nairobi and Mombasa are reported to be important transit points

for illicit drugs (UNODC, 2014).

Drug and substance abuse has been reported to exacerbate HIV/AIDS and TB pandemic

worldwide (Papas et al., 2011). Reports indicate that a considerable burden of HIV/AIDS

is attributable to drug abuse (Papas et al., 2011). For instance, injection drug use has been

associated with increased risk of disease transmission through needle sharing (Weber et

al., 2014). Similarly, non-injection drugs like alcohol have also been associated with
 3

heightened risk of HIV infection due to alteration of social behaviour (Kalichman et al.,

2007) when somebody is under the influence of alcohol. In Kenya, high prevalence of

hazardous drinking has been reported both in HIV infected and uninfected individuals

(Papas et al., 2011). This phenomenon has been linked to increased risky sexual

behaviour, poor adherence to anti-retroviral (ARV) drugs and the resultant ARV drug

toxicity among HIV infected patients (Kalichman et al., 2007). Furthermore, drugs and

substances have been reported to cause immune stimulation and induce inflammation

among the users hence hastening HIV symptoms (Kumar et al., 2012).

Cytokines, the proteins controlling inflammatory and immune-mediated reactions have

been involved in pathogenesis of HIV and TB co-infections (Diedrich and Flynn 2011).

Patients with HIV infection have altered cytokine balance, which has been reported to

exacerbate viral replication and susceptibility to opportunistic infections (Venketaraman

et al., 2011). For example, recent studies indicate that patients with acute HIV-1 infection

exhibit elevated plasma levels of anti-inflammatory cytokine, IL-10 and pro-

inflammatory cytokines, tumor necrosis factor (TNF)-, and IL-1 compared to uninfected

group (Roberts et al., 2010). Similarly, elevated TNF- and IL-10 levels have been

correlated with higher viral loads during the acute phase of HIV infection (Roberts et al.,

2010). These immunological alterations are reported to accelerate HIV/AIDS

pathogenesis among the affected patients subsequently increasing their susceptibility to

opportunistic infections (Roberts et al., 2010).

 4

Tuberculosis on the other hand is an inflammatory disease associated with production of

inflammatory mediators leading to cachexia, immunologic and metabolic derangements.

Patients with active pulmonary TB have been shown to exhibit elevated plasma levels of

TNF-, IFN-, IL-10 (Vignesh et al., 2013), and adiponectin (Keicho et al., 2012). Thus,

HIV-1 and TB co-infection has been suggested to enhance disease progression through

alteration of cytokine levels resulting in premature death of untreated patients

(Pawlowski et al., 2012).

Dysregulation in cytokine production has also been indicated among alcohol and drug

users. A recent study indicates that alcohol consumption has been associated with

increased plasma IFN- and IL-10 levels (Gonzale-Reimers et al., 2012). The use of

cigarettes (van Zyl-Smit et al., 2014) and bhang (Weiss et al., 2006) is reported to induce

IL-10 production but suppress IFN-. Since substance use is associated with impaired

cytokine production that also characterizes HIV and TB infections, determination of

cytokine levels in HIV and TB co-infection among substance users may be useful in

unveiling the immunologic interaction of these three major health challenges.

While previous studies have established immunologic derangements in HIV-1 and TB as

well as drug abuse in developed countries (Oliver et al., 2012; Neupane et al., 2014), the

data in developing nations is inconsistent and/or lacking (Kassa et al., 2013; Mihret et al.,

2014). In addition, the role of drugs and substances in HIV and TB disease progression is

largely unknown.
 5

1.2 Problem statement and justification

In Kenya, substance use is among the major social problems facing major towns

including Nairobi, Mombasa and Kisumu. Mombasa town a coastal city in Kenya is

reported to have the highest prevalence of injection drug use in the country (NACC,

2012). The prevalence of drug use and subsequent addiction is reported to be high among

HIV infected individuals (Kumar et al., 2012). While many studies have established

immunologic changes in mono-infected HIV and TB patients, immuno-pathogenesis of

these co-morbidities in the patient during concurrent infection in Africa is unknown

(Kassa et al 2013). Therefore, this cross-sectional study determined plasma levels of

some selected, clinically important cytokines including IFN-, IL-10 and adiponectin as

markers of disease progression in HIV and TB co-infected ART-exposed non-injection

drug users from Mombasa County, Kenya. It is envisaged that the data provided may be

useful in understanding HIV-1 and TB disease progression that may help in designing

early intervention measures to be applied during management of HIV-1 and TB co-

infection among substance users. The findings may also assist in optimizing HIV-1 and

TB co-treatment in Africa and improve the capacity to conduct clinical trials.

1.3 Research questions

i. What are circulating levels of IFN-, IL-10 and adiponectin among HIV-1 and TB

co-infected and uninfected non-IDUs?

ii. What is the association of plasma IFN-, IL-10 and adiponectin levels with CD4+

T cell counts, BMI and HIV-1 viral load in HIV and TB co-infected non-IDUs?
 6

1.4 Hypotheses

i. Plasma levels of IFN-, IL-10 and adiponectin are similar in HIV and TB co-

infected and uninfected non-IDUs.

ii. There is no association among plasma IFN-, IL-10 and adiponectin levels and

CD4 T cell counts, BMI and HIV-1 viral loadin HIV-1 and TB co-infected non-

IDUs.

1.5 Objectives

To determine circulating IFN-, IL-10 and adiponectin levels as markers of disease

progression in HIV-1 and TB co-infected non-IDUs from Mombasa, Kenya.

1.5.1 Specific objectives

i. To determine plasma levels of IFN-, IL-10 and adiponectin in HIV-1 and TB co-

infected and uninfected non-IDUs.

ii. To examine associations of plasma IFN-, IL-10 and adiponectin levels with BMI,

CD4+ T cell counts and HIV-1 viral load in HIV-1 and TB co-infected non-IDUs.
 7

CHAPTER TWO

LITERATURE REVIEW

2.1Burden of HIV/AIDS, tuberculosis and drug use

Human immunodeficiency virus (HIV) is a lentivirus that infects and destroys CD4+ T

cells, macrophages and dendritic cells resulting in a multi-system syndrome referred to as

acquired immunodeficiency syndrome (AIDS) (Barr-Sinoussi et al., 2013).

Mycobacterium tuberculosis (MTB), on the other hand, is a rod-shaped, non-spore

forming aerobic bacilli, which infects alveolar macrophages and dendritic cells leading to

pulmonary tuberculosis (Lartey et al., 2011).

Globally, over 75 million people are infected with HIV resulting in about 36 million

deaths since the discovery of the disease in early 1980s. Almost, 0.8% of the adults aged

between 15-49 years are living with HIV with variations between regions and countries

(UNAIDS, 2014). Sub-Saharan Africa is the most affected region accounting for 71 % of

the global HIV burden (UNAIDS, 2014). Recent reports indicate that nearly 5% of adults

in Africa are HIV positive. The prevalence of HIV is gender based with the prevalence in

young women being more than twice as high as in young men (UNAIDS, 2014). In

addition, the prevalence of HIV among adults in Kenya is higher than the global

estimates. In 2011, almost 5.6% of adults aged between 15-49 years were living with

HIV (Kimanga et al., 2014) with an estimated 62,000 AIDS-related deaths in the country

(UNAIDS, 2014). In addition, reports indicate that HIV infection rate is higher in urban

areas than in rural areas, although men in rural areas are more likely to be infected with
 8

the virus than those in urban areas (Kimanga et al., 2014). Therefore, there is need to

upscale HIV counselling and treatment both in rural and urban areas.

Globally, TB is the second leading cause of infectious disease burden after HIV (WHO,

2014). Approximately, 11 million new TB cases and over 1.5 million deaths occurred in

2013 (WHO, 2014). Like HIV, the prevalence of TB is high in Africa with almost 2.8

million people suffering from TB, contributing an estimated 29% of the total global TB

cases in 2013 (WHO, 2014). Kenya is among the 22 countries with over 80% of TB cases

worldwide, and is regarded as a high TB burdened country (WHO, 2014). In 2013, the

incidence rate of TB was approximately 253 cases per 100,000 populations, ranking the

country at position eleven among highly burdened countries (WHO, 2014). Even though

there has been a general decline in TB mortality rate of 45% from 1990-2013 globally,

the African region is far from meeting the Stop TB partnership target that espouses

halving mortality by the current year (UNAIDS, 2014). Thus, African countries should

upscale the prevention, diagnosis and treatment of TB to halt the disease pandemic in the

region.

HIV and TB infections have a synergistic interaction, fuelling progression of one another.

Recent reports indicate that almost 13% of an estimated 9 million people who developed

TB worldwide in 2013 were HIV-1 positive with 78% residing in Africa (WHO, 2014).

Kenya has been classified as a high-burden country for the dual epidemic with over 30%

of incident TB cases being attributable to HIV in 2012 (Yuen et al., 2014). It is reported
 9

that TB aggravates HIV infection. For example, recent studies in Kenya indicate that

almost 33% of persons with TB are infected with HIV compared to 5.1% of persons

without TB (Mbithi et al., 2014). The prevalence of TB-related deaths among HIV

patients is still high in the country despite implementation of the WHO policy of ART

initiation to all HIV and TB co-infected patients (Mbithi et al., 2014). This calls for an

increased HIV testing and care facilities to halt HIV/AIDS epidemic.

Drug and substance use is a global health and social challenge. It is estimated that over

200 million adults used an illicit drug with almost 183,000 drug-related deaths occurring

worldwide in 2012 (UNODC, 2014). The extent of drug use varies by region, type of

drug used and gender. It is reported that men are three-fold more likely to use an illicit

substance compared to women (UNODC, 2014). Regionally, the prevalence of bhang,

opiods, opiates and amphetamines use is reported to be highest in America (UNODC,

2014). Currently, the information on the burden of drug use in African region is limited.

Recent surveys in West and central Africa reveal an increasing trend in bhang and

cocaine use but reduced overall use of other substances like heroin and amphetamines

(UNODC, 2014). Various drugs and substances are associated with major health

conditions including addiction, lung and mental problems, poor psychosocial

development and increased risk of HIV infection (UNODC, 2014).

 10

2.2 Pathogenesis of HIV

The clinical course of HIV infection constitutes three phases including the acute phase,

the chronic infection and the AIDS-defining illness. Early HIV infection induces

production of antibodies against viral proteins as well as enhanced cytokine responses to

contain the invading virus. The acute phase, also called primary phase is characterized by

increased viremia (up to 107 copies of viral RNA per millilitre of blood) and high number

of infected CD4+ T cells in blood and lymph nodes (Coffin and Swanstrom, 2013). The

acute phase is followed by chronic infection, also known as a period of clinical latency.

The chronic HIV infection stage is characterized by decreased viral replication, stable or

slowly diminishing numbers of CD4+ T helper cells and defects in T cell function

(Coffin and Swanstrom, 2013). Finally, the AIDS-defining illness sets in when the CD4+

T cell count falls below 200 cells/mm3.

It has been suggested that HIV infection destroys CD4+ T cells via three main

mechanisms; through direct killing of the infected cells, increased apoptosis of the

infected cells and through indirect killing of infected cells by CTLs (Barr-Sinoussi et al.,

2013). Since HIV infection depletes CD4+ T cells, enumeration of these cells has been an

important surrogate for HIV pathogenesis (Coffin and Swanstrom, 2013). In addition,

discovery of ART has also relied on CD4+ T cell counts in terms of initiation period and

the patients response to HIV therapy. The world health organization recommends that

HIV-infected individuals with CD4+ T counts 320 cells/ml of blood should be initiated

on ART. This is because a low CD4 cell count has been associated with treatment failure
 11

and increased mortality (Wejse et al., 2013). A critical value of CD4+ T cell counts 200

cells/ml has hence been set for diagnosis of AIDs (Kassa et al., 2013). In HIV and TB co-

infection, lower levels of CD4+ T cells have been considered a risk factor for progression

of latent TB to active TB (Coffin and Swanstrom, 2013) and for occurrence of immune

reconstitution inflammatory syndrome (IRIS) during the initiation of treatment in

combined ART-naive patients (Kassa et al., 2013).

2.3 Monitoring of HIV/AIDS disease progression

Immunological and clinical monitoring are the most common criteria for predicting the

course of HIV infection (da Silva et al., 2013). However, routine determination of HIV

RNA levels has recently been recommended for monitoring disease progression in HIV

infected patients (Gupta et al., 2009). In addition, markers of immune activation have

also been suggested to predict HIV progression (da Silva et al., 2013). These markers

include the determination of levels of 2-microglobulin, neopterin, antibodies such as

anti-p24, anti-gp120, anti-p17, anti-gp41, anti-nef, anti-CD4, as well as the anti-leucocyte

antibodies (Oliver et al., 2010). Furthermore, the plasma levels of cytokines such as

TNF-, interferons and IL-2 have also been used as markers of disease progression in

HIV patients (Oliver et al., 2010).

 12

2.3.1 HIV-1 viral loads in HIV-1/AIDS

HIV-1 viral load measurement indicates the number of copies of HIV-1 genomic RNA

per millilitre of plasma. Viral load is an important tool for diagnosis and prognosis of

HIV-1 infection. A prospective study in gay men has revealed an inverse correlation

between the level of viremia and the time of disease progression to AIDS (Coffin and

Swanstrom, 2013). In this study, men with higher HIV-1 viral loads progressed to AIDS

four times faster compared to those with reduced plasma viral copies (Coffin and

Swanstrom, 2013). Therefore, HIV particle levels in plasma can provide a good measure

of the rate at which the viral infection damages the immune system of the infected

patient.

Plasma viral loads are also used to monitor the efficiency of ARV drugs, whereby, ability

of an ARV drug to halt or even reverse progression of HIV-1 infected patient to AIDS

correlates with its ability to suppress viremia (Coffin and Swanstrom, 2013).

Consequently, WHO has recommended viral load monitoring as a criterion for

monitoring response to ARV treatment and diagnosis of failed treatment in HIV-1

infected patients (Petersen et al., 2014). Treatment failure is indicated by the presence of

persistently detectable viral load exceeding 1000 copies/ml of blood (Petersen et al.,

2014). As a result, it has been suggested that viral load should be determined within six

months following ART-initiation and then at least after every 12 months to detect any

treatment failure (Petersen et al., 2014). Routine checking of HIV-1 viral load during

HIV-1 infection is preferred to immunologic and clinical monitoring because it provides

 13

an early and more accurate indication of treatment failure (Boulle et al., 2013).

Therefore, viral load monitoring may determine the need to switch to second line drugs,

thus reducing the accumulation of drug-resistance mutations thereby improving clinical

outcomes (Boulle et al., 2013). Moreover, it has been established that measuring viral

load can help distinguish between treatment failure and non-adherence, hence may also

predict the risk of disease transmission at the population level (Petersen et al., 2014).

Clinical studies have revealed that the risk of HIV-1 transmission and progression is very

low when the viral load is lower than 1000 copies/ml (Boulle et al., 2013).

2.4Tuberculosis pathogenesis

Mycobacterium tuberculosis (MTB) is transmitted by inhalation of droplet nuclei

expelled through coughing, sneezing, speaking or singing from a person with pulmonary

or laryngeal TB (Knechel, 2009). Once inhaled, the bacilli travel to the alveoli where

they cause pulmonary TB, although the bacteria can also spread to other organs such as

lymphatics, bones, spine or kidneys and cause extra-pulmonary TB (Knechel, 2009).

Studies have reported that alveolar macrophages engulf and degrade mycobacteria in the

lungs by production of proteolytic enzymes and cytokines (Knechel, 2009). However, the

bacilli can continue to multiply within the macrophages and are released when the

macrophages die (Knechel, 2009). In immuno-competent persons with intact cell-

mediated immunity, nodular-like lesions called granulomas usually form around the

bacteria (Knechel, 2009). The granulomas are formed from accumulation of activated T

lymphocytes and macrophages and create a micro-environment that limits multiplication

 14

and spread of mycobacteria (Elliott et al., 2009). In such individuals, the viable bacilli

can remain enclosed within the lesions for years or even lifetime, a state called latent TB.

Persons with latent TB are asymptomatic and not infectious, however, latent TB can be

reactivated if the immune system becomes compromised (Knechel, 2009).

2.5 Body mass index in HIV and tuberculosis

Body mass index (BMI), an anthropometric indicator calculated from weight and height

characterize nutritional status of populations. It is generally regarded that individuals with

BMI less than 18.5 are underweight, those between 18.5 and 24.9 are normal while those

with BMI above 25 are overweight or obese (Mariz et al., 2011). In addition to viral and

immunologic measures, long term monitoring of BMI has also been used to predict HIV-

1 disease progression in HIV infected patients (Langford et al., 2007). Furthermore, a

low BMI (<18.5kg/m2) is an established marker of poor prognosis in HIV patients and

has also been associated with increased risk of TB reactivation (Hanrahan et al., 2010).

On the other hand, decreased BMI has also been associated with increased mortality rate

among HIV and TB co-infected patients (Benova et al., 2012). These studies hence

suggest that BMI measurement can be utilized as a valuable surrogate marker for HIV

and TB disease progression.

2.6 Role of cytokines in HIV and tuberculosis pathogenesis

Dysregulation in cytokine production and the ensuing inflammation characterize both

HIV and TB infections. It has been reported that during the course of HIV infection there
 15

is an initial rise in the levels of pro-inflammatory cytokines such as TNF- and IFN-

followed by anti-inflammatory cytokines such as IL-4 and IL-10 (Stacey et al., 2009).

This may suggest that during acute HIV infection, pro-inflammatory cytokines are

produced to contain the virus followed by anti-inflammatory cytokines to modulate the

immune reactions (Stacey et al., 2009). However, recent studies have shown discordant

roles of cytokine groups during HIV-1 infection (Roberts et al., 2010). Among pro-

inflammatory cytokines, IL-7 and IL-15 have been associated with higher viral set points

while IFN- and IL-12 have been correlated with lower set point (Roberts et al., 2010).

Viral set points have been used to predict the course of HIV-1 infection, with higher viral

set points being associated with greater HIV-1 pathogenesis and mortality (Katsikis et al.,

2011). Surprisingly, higher plasma IL-10 levels have also been associated with protection

against HIV-1 infection (Katsikis et al., 2011). Therefore, even though immune response

protects the body against invading pathogens severe immune activation may result in

inflammation promoting disease progression.

Similarly, cytokine dysregulation characterizesTB resulting in impaired immune response

(Tadokera et al., 2011). Pro-inflammatory cytokines TNF- and IFN- play a key role in

granuloma formation that controls mycobacterial replication, maintaining it in latent state

(Diedrich and Flynn, 2011). In addition, TB products induce increased expression of

chemokine receptors CXCR4 and CCR5 on infected cells (Pawlowski et al., 2012).

However, cytokines induced by TB infection have been shown to promote HIV

pathogenesis (Pawlowski et al., 2012). For instance, TNF- has been linked to increased
 16

viral replication in macrophages, while CXCR4 and CCR5 are co-receptors for HIV thus

promote entry of the virus into host cells (Pawlowski et al., 2012).

In addition to cytokines produced by immune cells, other cytokines particularly those

secreted by the adipose tissue have also been found to affect the immune response to HIV

infection. For example, it is reported that adiponectin inhibits T-cell proliferation (Koethe

et al., 2013) and correlates inverselywith CD4+ T cell counts in HIV-positive patients on

ART (Kosmiski et al., 2008). Additionally, low adiponectin levels have been revealed in

HIV-1 positive patients relative to healthy individuals (Koethe et al., 2013). Therefore,

determination of cytokine levels in HIV-1 infected patients may be a valuable surrogate

for monitoring HIV-1 disease progression.

2.6.1 Interferon-gamma in HIV and tuberculosis pathogenesis

Interferon-gamma is a dimeric protein produced by natural killer (NK) cells, CD4+ Th1

cells and CD8+ T cells in response to the presence of both bacterial and viral antigens in

blood plasma. Unlike type-1 interferons (IFN- and IFN-), IFN- has no direct antiviral

activity (Roff et al., 2014). It is reported that IFN- stimulates production of IL-12 in

activated antigen presenting cells (APCs) thus amplifying Th1 response while

suppressing Th2 responses (Roff et al., 2014). In addition, IFN- enhances expression of

MHC class-II molecules on APCs thus enhancing their antigen presenting ability to CTLs

for destruction. On the other hand, HIV-1 transactivator protein when bound to its
 17

receptor interferes with IFN- intracellular signalling pathways, thus lowering the antigen

presenting capacity of the APCs (Roff et al., 2014).

Studies determining the effects of combined ART on IFN- levels have shown

contradicting results. Earlier longitudinal studies in HIV-1 infected adults revealed steady

increase of IFN- levels throughout the acute stage of HIV infection but the plasma levels

remained high even on introduction of ART (Watanabe et al., 2010). However, in a

recent study, untreated HIV-1 patients had higher levels of IFN-, which markedly

decreased on initiation of combined ART (Malherbe et al., 2014). Some investigators

have also hypothesized that combined-ART up-regulates circulating IFN-, which has

been associated with immune reconstitution inflammatory syndrome in HIV-1/AIDS

patients (Zheng et al., 2014). Therefore, there is need to further investigate the role of

IFN- in HIV-1 pathogenesis particularly during administration of combined therapy.

Interferon- also plays a key role in pathogenesis of TB.Mycobacterium

tuberculosisactivates the immune cells to secrete IFN- that assists in controlling its

spread (Pawlowski et al., 2012). Studies by Green et al (2013) showed that mice deficient

of IFN- gene were more susceptible to TB and died earlier than the wild type mice.

Higher plasma IFN- levels have been associated with severe pulmonary and extra-

pulmonary TB disease (Abebe, 2012). Quantitative determination of IFN- has been used

as a diagnostic tool for active TB (Trajman et al., 2013) as well as monitoring its

treatment (Abebe, 2012).

 18

Both HIV-1 and TB infections stimulate release of IFN- from T cells. Similar results

have been demonstrated by a recent Ethiopian study involving HIV and TB mono- and

co-infected individuals (Mihret et al., 2014). However, earlier studies hypothesized that

both HIV-1 and TB infections can inhibit T-cell effector functions such as the production

of IFN- and that co-infection is associated with more profound suppression of type 1

responses (Geldmacher et al., 2010). For instance, peripheral blood mononuclear cells

(PBMCs) from HIV-1 infected patients with latent TB stimulated with MTB lysate

expressed less IFN- than PBMC from HIV-1 negative latent TB patients (Mendona et

al., 2007). Therefore, co-infection with HIV-1 and TB results in dysregulation of IFN-

levels which may interfere with immunological responses against these infections in co-

infected patients.

2.6.2 Role of interleukin-10 in HIV and tuberculosis pathogenesis

Interleukin-10 is an anti-inflammatory cytokine that controls innate immune reactions

and cell mediated immunity (Porichis and Kaufmann, 2011). Since both HIV-1 and TB

are associated with chronic inflammation, IL-10 is reported to play a crucial role in

prognosis of these pathogens in the HIV and TB infected patients (Porichis and

Kaufmann, 2011). Studies on the role of IL-10 in HIV-1 pathogenesis show conflicting

results. It has been proposed that different stages of the HIV-1 disease determine the role

that IL-10 plays in HIV infected patients (Naicker et al., 2009). Elevated IL-10 levels

have been reported to promote viral replication during acute HIV infection while

suppressing HIV replication in macrophages during chronic disease (Naicker et al.,

 19

2009). Recent studies have associated elevated plasma IL-10 levels with viral loads and

greater risk of CD4+ T cell loss during acute HIV disease (Roberts et al., 2010; Liu et al.,

2014), suggesting that HIV-1 infection induces early IL-10 production as an adaptive

mechanism to evade the patients immune responses.

In contrast to Naicker et al., (2009), similar studies have revealed adverse effects of

elevated circulating IL-10 levels in chronic HIV-1 patients (Brockman et al., 2009).

Patients with persistent viral replication are reported to have elevated plasma IL-10 levels

compared to those with undetectable viral particles and healthy individuals (Brockman et

al., 2009). Circulating IL-10 levels have been positively correlated with HIV-1 viral

loads and successful ARV treatment is reported to reduce plasma IL-10 levels (Brockman

et al., 2009). Thus, the viral antigen could be the main driver of increased IL-10 levels

during chronic HIV-1 disease.

Interleukin-10 can inhibit immune response to MTB and may hence, contribute to

progressive TB disease (Redford et al., 2011). A recent study has revealed elevated IL-10

levels in the lungs and serum of active pulmonary TB patients (Redford et al., 2011).

Furthermore, in vitro blockade of IL-10 pathways is reported to increase IFN- secretion

and enhance phagosomes maturation in MTB-infected macrophages (OLeary et al.,

2011). Thus, elevated IL-10 levels observed during TB infection could be a survival

mechanism for MTB.

 20

Studies on IL-10 levels in HIV and TB co-infection have reported conflicting results

(Benjamin et al., 2013; Tomlinson et al., 2014). In studies using plasma samples, HIV-1

and TB co-infected patients are reported with elevated plasma IL-10 levels (Benjamin et

al., 2013) that normalize on introduction of an effective combined ART and TB therapy

(da Silva et al., 2013). Conversely, a recent study using saliva and brocho-alvoelar lavage

fluid samples has reported reduced IL-10 levels in HIV and TB co-infected patients

compared to TB-negative individuals (Tomlinson et al., 2014). Therefore, this call for

further research to ascertain the arising differences and to understand better the role of

IL-10 in HIV and TB pathogenesis.

2.6.3 Role of adiponectin levels in HIVand TB pathogenesis

Adiponectin, also known as adipocyte complement-related protein of 30 kDa (Acrp30),

adipoQ, adipose most abundant gene transcript 1 (apM1) or gelatin-binding protein of 28

kDa (GBP28), is predominantly secreted by adipocytes but also by skeletal muscles,

endothelial cells, cardiomyocytes, lymphocytes and placenta (Robinson et al., 2011).

Adiponectin primary structure consists of 244 amino acid protein encoded by apM1 gene

with a structure similar to collagen VII and X and complement factor C1q (Adamczak

and Wiecek, 2013). The plasma levels of adiponectin are reported to range from 2-30

g/mL, and are generally higher in females than males (Bidulescu et al., 2013). The

production of adiponectin is inhibited by pro-inflammatory factors such as TNF- and

IL-6 as well as hypoxia and oxidative stress (Robinson et al., 2011). Adiponectin effects

include insulin sensitivity, increased glucose uptake and decreased gluconeogenesis,

 21

stimulation of -oxidation and triacylglycerol clearance, weight loss, protection from

endothelial dysfunction, control of energy metabolism, and anti-inflammation (Robinson

et al., 2011).

Adiponectin levels negatively correlate with BMI and waist-hip circumference. Thus,

reduced plasma adiponectin has been associated with obesity, type-2 diabetes,

cardiovascular disease and HIV-lipodystrophy (Robinson et al., 2011). In HIV-

lipodystrophy, serum adiponectin levels are lower in patients with fat redistribution and

correlate negatively with LDL-cholesterol, total cholesterol, triacylglycerols, glucose and

insulin resistance but positively with HDL-cholesterol (Zinn et al., 2013). Thus, low

adiponectin levels are a risk factor for cardiovascular disease, type-2 diabetes and

metabolic dysfunction in HIV-1 patients.

Although circulating levels of adiponectin in HIV-1 and other inflammatory diseases like

obesity and type-2 diabetes are clear, the results in TB are contradictory. Previous studies

in China demonstrated lower adiponectin levels in active pulmonary TB patients relative

to latent TB or the healthy controls (Xu et al., 2007). However, recent studies (Santucci

et al., 2011; Keicho et al., 2012), established that high adiponectin levels were associated

with severe pulmonary TB. Thus, further studies have been suggested to understand the

role of adiponectin in TB and explain the arising discrepancies (Yurt et al., 2013). In

addition, there is no data on circulating adiponectin levels in HIV/TB co-infection.

 22

2.7 Drug use and cytokine dysregulations

Drug and substance use is associated with dysregulation in cytokine production and

inflammation. For instance, alcohol is reported to induce increased plasma IFN- and IL-

10 levels (Gonzalez-Reimers et al., 2012) that have been associated with cirrhosis among

active drinkers. Bhang on the other hand has been reported to suppress production of

IFN- while inducing IL-10 production (Weiss et al., 2006), hence it may lead to

immuno-suppression. In vitro studies have also shown that cigarette smoke exposure

reduces IFN- production by the lung CD4+ T-cells (Feng et al., 2011) and plasma IFN-

levels are increased following cigarette smoking cessation (Shaler et al., 2013).

Drugs and substances are also reported to influence expression and production of

adiponectin (Kotani et al., 2012; Won et al., 2014). Previous studies have reported

reduced plasma adiponectin levels among cigarette smokers compared to non-smokers

(Tsai et al., 2011). It is suggested that nicotine, an active component of cigarette smoke

inhibits expression of adiponectin gene in adipocytes (Kotani et al., 2012). In addition,

smoking may induce oxidative stress and inflammatory cytokines like TNF- that is

known to inhibit expression of adiponectin gene in adipose cells (Kotani et al., 2012).

Alcohol consumption has also been associated with reduced plasma adiponectin levels

(Jung et al., 2013) by inhibiting expression of peroxisome proliferator activated receptor

that is involved in synthesis of adiponectin (Kotani et al., 2011). It is reported that

cessation from smoking and alcohol use increases plasma levels of adiponectin (Jung et

al., 2013; Won et al., 2014). Therefore, smoking and alcohol cessation can prevent
 23

metabolic disorders associated with reduced adiponectin by stimulating synthesis of

adiponectin from adipocytes.

Since various drugs and substances cause cytokine dyregulations, concomitant use of

drugs during HIV and TB may result in increased morbidity and mortality among the

patients. Therefore, HIV and TB patients should be discouraged from drug abuse to

prevent undesirable effects of these drugs.

 24

CHAPTER THREE

MATERIALS AND METHODS

3.1 Study area

The study participants were recruited at Bomu hospital, a social enterprise facility, in

Mombasa County (Appendix IIV). Mombasa County is located at the coastal region of

Kenya and covers an area of approximately 212.5 km2 with a population of almost

900,000 people according to 2009 census report (KNBS, 2010). The county is Kenyas

major tourist attraction centre and trade hub (Valle & Yobesia, 2009). However, there is

high poverty index level of almost 37.6% in the county (Kristjanson et al., 2010),

partially attributed to high HIV/AIDS prevalence rate. For example, by the end of 2011,

almost 77,000 people in Mombasa were reported to be living with the HIV (NACC,

2012). Moreover, the county is known for high incidences of HIV-risky behaviours

including injection and non-injection drug use, homosexuality and commercial sex work

(NACC, 2012), all of which are known predisposing factors for high HIV-1 infection

rates (Weber et al., 2014).

3.2 Study design

This study utilised an experimental laboratory-based cross-sectional design to determine

circulating IFN-, IL-10 and adiponectin and their association with CD4+ T cell counts,

HIV RNA copies and BMI in HIV and TB co-infected non-injection substance users

recruited at Bomu hospital in Mombasa County.

 25

3.3 Ethical considerations

This study was part of a larger study investigating the determinants of injection and non-

injection substance use in Mombasa County that was approved by Kenyatta University

Ethical Review Committee (Appendix I). Written informed consent was obtained from

each participant prior to his or her enrolment in to the study (Appendix II). All the study

blood samples were obtained by qualified phlebotomists, coded and the results handled

confidentially. The participants benefited from free health education on HIV, TB,

sexually transmitted infections, hygiene and nutrition.

3.4 Sample size calculation

The size of samples collected was based on (Dallal, 2012) formula:

n = [162/2] +1

n = [16 303.142/ (809.78 - 87.88)2] + 1

n=4

Where,

n is the desired sample size

is the standard deviation

is the mean difference

The sample size was calculated based on previous studies showing mean plasma IFN-

levels of 87.88 pg/ml in HIV-1 and TB co-infected patients and 809.78 pg/ml in TB

mono-infected (Adewole et al., 2013) were used in calculating the sample size. From this
 26

study the mean difference () in plasma IFN- levels between the TB mono-infected

and the HIV/TB co-infected groups was 721.98 pg/ml. In addition, the standard deviation

in the mean plasma IFN- levels were 476.28 pg/ml in the mono-infected and 130.0

pg/ml in the co-infected patients giving a mean value of 303.14 pg/ml (Adewole et al.,

2013).

Therefore, substituting these values in the formula above gave a desired minimum sample

size of at least 4 subjects in each of the study groups. Thus, the following individuals

were recruited into each of the study groups: HIV-1 and TB co-infected antiretroviral

treatment (ART)-naive (HIV-1[+]/ART[-]/TB[+], n=12) and exposed (HIV-1

[+]/ART[+]/TB[+], n=21); HIV-1 mono-infected ART-naive (HIV-1 [+]/ART[-]/TB[-],

n=6) and exposed (HIV-1 [+]/ART[+]/TB[-], n=9); TB mono-infected (HIV-1 [-

]/TB[+], n=5); and uninfected (HIV-1 [-]/TB[-] non-IDUs, n=32); and healthy controls

(HC, n=27).

3.5 Study population

The target population included HIV and TB co-infected non- IDUs from Mombasa

County while the study population consisted of the following study groups: HIV-

1[+]/ART[-]/TB[+],n=12; HIV-1 [+]/ART[+]/TB[+], n=21; HIV-1 [+]/ART[-]/TB[-],

n=6; HIV-1 [+]/ART[+]/TB[-], n=9; HIV-1 [-]/TB[+], n=5; and uninfected HIV-1 [-

]/TB[-] non-IDUs, n=32; and HC, n=27.

 27

3.6 Inclusion and exclusion criteria

Only consenting adults aged 18 years and above were enrolled in to the study. All the

non-IDUs recruited had used at least one non-injection drug as classified by UNODC

(2014), a month prior to the study. In addition, healthy controls were recruited among

individuals who were not using any drug and substance as defined by UNODC (2014)

and were HIV-1-negativeand TB negative.However, candidates using injection drugs,

pregnant women and those below 18 years were excluded from the study.

3.7 Demographic data collection

Demographic information and anthropometric measures including age, gender, ART use

height, weight, body temperature, and drug use were obtained for each participant at

enrolment. Data was captured using data forms (Appendix III).

3.8 HIV-1 diagnosis

HIV testing was performed on whole blood samples obtained by finger-prick at

enrolment. Rapid serologic tests, Determine (Abbott Laboratories, Tokyo, Japan) and

Unigold (Trinity Biotech Plc, Bray, Ireland) were used for testing HIV-1 infection.

Briefly, 250 l of blood was collected in an EDTA vacutainer tube (BD, Franklin Lakes,

USA) then placed in a sample port for testing. The wash buffer was added followed by

incubation for 10 minutes. The test results were read and recorded. Study participants

with positive results for both Determine and Unigold were considered HIV infected.
 28

3.9 Tuberculosis screening and diagnosis

Tuberculosis screening and diagnosis was performed by qualified and experienced

clinicians, and laboratory technologists. Laboratory tests were performed on sputum

samples obtained from study participants. Five ml of sputum samples were collected for

three different consecutive days in a sputum collection booth (model VCM-1500N2).

This was followed by placing the most mucoid part of the sputum on the slide in an oval

shape using an applicator stick. Carbol fuchsin dye was then applied on the slide, which

was then heated gently until it steamed. The hot slide was allowed to rest for 5 minutes to

fix the dye and afterwards rinsed with tap water. The decolouriser (3% hydrochloric acid

in isopropyl alcohol) was then added to the slide and rinsed using tap water. Next, the

slide was filled with methylene blue, rinsed with tap water and blotted. The non-acid-fast

bacteria picked up methylene blue, to become blue while the acid-fast bacteria (AFB)

retained carbol fuchsin colour and appeared red when viewed under the microscope. A

drop of oil was placed at one end of the smear and the slide was viewed under oil

immersion lens.

Pulmonary TB case was identified based on the presence of at least one acid fast bacilli

(AFB+) as per WHO recommendations (WHO, 2014). The results were reported as

negative where no AFB was found in at least 100 fields and exact figure out of 100 in

slides with 19 AFB per 100 fields. The slides with 1099 AFB per 100 fields were

reported as positive (+). However, slides with 110 AFB per field with a count of at least

50 fields were reported as (++) while slides with more than 10 AFB per field with counts
 29

of at least 20 fields were reported as (+++). The patients who tested TB positive were

enrolled into the TB programme at Bomu hospital for treatment. Infectious waste was

autoclaved then burned.

3.10 Measurement of body mass index (BMI)

The height (m) of each enlisted candidate was measured to the nearest cm using the

Health-O-meter PORTROD wall mounted height rod (Healthometer, McCOOK, USA)

while body weights (kg) were measured to the nearest 1.0 grams using Feet design

standard electronic manual weighing scale (Richforth Electronics Co., Fuzhou, China).

Body mass index was calculated using the height (m) and weight (kg) measurements of

the study participants using the formula:

BMI = [Weight (kg) Height (m) 2] (Dessinioti and Zouboulis, 2014).

3.11 Blood collection and processing

Venipuncture was performed using a 10 ml needle and a G21 syringe. A tourniquet was

placed on the upper arm to distend the veins, and the medial vein was punctured and

about 10 ml of blood collected in to the syringe. The blood samples were then transferred

into purple top ethylenediaminetetraacetic acid (EDTA) BD vacutainer tubes (BD,

Franklin Lakes, USA). Plasma samples were obtained by centrifuging at 2000 revolutions

per minute at 40C for 20 minutes then aspirating the upper layer and transferring the

aliquots into 1.5 ml labelled eppendorf tubes. The tubes were then transferred into freezer

boxes then stored at -80C until used for cytokine measurements and viral load analysis.
 30

3.11.1 Determination of HIV-1 viral load

Abbot m200 system was used to determine HIV-1 viral loads according to the

manufacturers instructions (Abbott Molecular Inc., Illinois, USA). The system has two

instruments: the Abbott m2000sp system for automated extraction, purification and

preparation of RNA from the samples and m2000rt, a real-time polymerase chain

reaction instrument for amplification and detection of HIV. Briefly, samples were loaded

into the machine followed by RNA extraction from 0.2 ml plasma samples using the

Abbott m2000sp system. The master mix containing the viral RNA was then transferred

to the Abbott m2000rt instrument for viral load detection at a lower limit of viral load

quantification of 150 (2.18 log10) copies/mL of blood. Fluorescence release of the HIV-1

probe was proportional to the amount of HIV-1 target sequence in the sample. The

fluorescence intensity of HIV-1 probe was converted into viral loads by the analyzer.

3.11.2 CD4+ T cell measurements

Baseline CD4+ T cell counts were determined using a FACSCalibur flow cytometer

(Becton-Dickinson, NJ) equipped with automated acquisition and analysis software

according to the manufaturers instructions. Briefly, 50lwhole blood sampleswere added

into reagent tube containing BD Tritest CD3 fluorescein isothiocyanate (FITC), CD4

phycoerythrin (PE) and CD45 peri-dinin chlorophyll protein (PerCP) fluorescent-tagged

monoclonal antibodies.Samples werethen vortexed and incubated for 15 minutes. Fifty

(50) l fixative solution wasthen addedand the samples analysed. The absolute CD4+ T
 31

cell counts and the corresponding percentages were determined automatically by in-built

Attractors software programme.

3.11.3 Measurement of plasma interferon-gamma

Circulating IFN- levels in the samples was determined using a sandwich ELISA kit

according to the manufacturers protocol (eBioscience, San Diego, USA). Briefly,

ELISA microtitre plate wells (MaxiSorp, Nunc International, Denmark) were coated

with 100l of mouse anti-human IFN- capture antibody (R&D SystemsEurope, UK)

diluted in phosphate buffered saline (PBS), pH 7.4 (137 mM sodium chloride, 2.7 mM

potassium chloride, 10 mM sodium phosphate dibasic and 1.8mM potassium phosphate

monobasic). The plates were then sealed with parafilm and incubated overnight at 4oC.

Plate wells were then aspirated and washed 3 times using 250l of wash buffer

containing 0.05% Tween 20 in PBS. The plates were blocked by adding 300L of

blocking buffer containing 0.1% bovine serum albumin (BSA), 0.05% Tween 20 in Tris

buffered saline followed by incubation at room temperature for 1 hour. The plates were

washed and blotted then 100l of standards and samples diluted in assay buffer added

into appropriate wells. Plates were then covered and incubated at room temperature for

two hours. The solutions were discarded, and the wells washed 3 times using wash

buffer. After the last wash, 100l of detection antibody (biotin-conjugated secondary

antibody) was added to the plates, sealed and incubated at room temperature for 1 hour.

The plates were then washed as earlier described and 100l of detection enzyme

(streptavidin-horse radish peroxidase (HRP) was then added to each plate well, and the
 32

plates incubated at room temperature for another 20 minutes. The wash step was repeated

and 100l of substrate solution (tetramethylbenzidine, TMB) added to each plate well

and incubated for 20 minutes at room temperature. Fifty l of stop solution (2N H 2SO4)

was then added to each plate well to stop the reaction. The absorbance (optical densities,

OD) of each well was read at a wavelength of 450 nm with a reference wavelength of 630

nm using the Dynatech MR5000 Microplate Reader (Dynex Technologies Inc.,

Sullyfield, USA). Sample concentrations were calculated using the appropriate standard

calibration curves (1000 500 250 125 62.5 31.25 15.62 and 0.00 pg/ml) of the

corresponding recombinant human IFN- protein included in each assay plate.

3.11.4 Determination of plasma Interleukin-10 levels

The circulating IL-10 levels in the study samples were determined using a sandwich

ELISA kit according to the manufacturers protocol (eBioscience, San Diego, USA).

Briefly, ELISA microtitre plates (MaxiSorp, Nunc International, Denmark) were coated

with 100l of mouse anti-human IL-10 capture antibody (R &D Systems Europe, UK)

diluted in PBS. The plates were then sealed with parafilm and incubated overnight at 4 oC.

Plate wells were aspirated and washed 3 times using 250l of wash buffer containing

0.05% Tween 20 in PBS. The plates were then blocked by adding 300L of blocking

buffer containing 0.1% BSA, 0.05% Tween 20 in Tris buffered saline followed by

incubation at room temperature for 1 hour. The plates were washed, blotted then 100l of

standards and samples diluted in assay buffer added into appropriate wells. Plates were

then covered and incubated at room temperature for two hours. The solutions were
 33

discarded, and then the wells washed 3 times using the wash buffer. After the last wash,

100l of detection antibody (biotin conjugated secondary antibody) was added, plates

sealed and incubated at room temperature for 1 hour. The plates were washed and 100l

of detection enzyme (streptavidin-HRP) added to each plate well and the plates incubated

at room temperature for another 20 minutes. The wash step was repeated and 100l of

substrate solution (TMB) added to each plate well and incubated for 20 minutes at room

temperature. Fifty l of stop solution (2N H2SO4) was then added to each plate well to

stop the reaction. Finally, optical density of each well was read at a wavelength of 450

nm with a reference wavelength of 630 nm using the Dynatech MR5000 Microplate

Reader (Dynex Technologies Inc., Sullyfield, USA). Sample concentrations were

calculated using the appropriate standard calibration curves (2,000 1,000 500 250

125 62.2 31.25 and 0.0 pg/ml) of the corresponding recombinant human IL-10 protein

included in each assay plate.

3.11.5 Measurement of plasma adiponectin levels

Plasma adiponectin levels were analyzed in samples using a sandwich ELISA kit

according to the manufacturers protocols (R&D Systems Europe, UK). Briefly, 100L

of anti-human adiponectin capture antibody (R&D Systems Europe, UK) was coated to

ELISA plates (MaxiSorp, Nunc International, Denmark). The plates were then sealed

with parafilm and incubated overnight at 4oC. The wells were aspirated and washed 3

times using 150 l of wash buffer containing 0.05% Tween-20 in PBS and 0.5% bovine

serum albumin (BSA). This was followed by blocking the wells using 300 l of blocking
 34

buffer (containing 0.5% BSA, 0.05% Tween-20 in Tris buffered saline) then incubating

for 1 hour at room temperature. The plates were washed, blotted and 100 l of samples

and standards diluted in assay buffer added into appropriate wells. The plates were

covered with parafilm and incubated for 1 hour at room temperature. The solution was

discarded and the wells washed 4 times then blotted on paper towels. A hundred L of

biotin-conjugated secondary antibody, and 100 L of detection enzyme streptavidin-HRP

added to each well and the plates incubated for 1 hour at room temperature. The solution

was then discarded and the wells washed. One hundred L of TMB substrate was then

added to each well and incubated for 30 minutes at room temperature. The enzyme HRP

acted on TMB substrate to produce a deep blue solution. Fifty L of the stop solution (2N

H2SO4) was added to each well to stop the reaction. On addition of a stop solution, the

deep blue solution turned yellow at the end of the reaction. The absorbance (optical

densities, OD) of each well was read at a wavelength of 450 nm with a reference

wavelength of 630 nm using the Dynatech MR5000 Microplate Reader (Dynex

Technologies Inc., Sullyfield, USA). Sample concentrations were calculated using the

appropriate standard calibration curves (18,000 - 6,000 - 2,000 - 666.7 - 222.2 - 74.1 -

24.7 and 0.0 ng/ml) of the corresponding recombinant human adiponectin protein

included in each assay plate.

3.12 Data processing and analysis

The raw data obtained was dual entered, cleaned and coded in excel spreadsheets (MS

Office) and exported into graph pad prism software for statistical analyses. Since the data
 35

was not normalised, non-parametric tests were used in statistical analyses. Categorical

data (gender and substance use) were summarized as proportions while continuous data

(age, anthropometric measures, CD4+ T-cell counts and HIV-1 viral load) were presented

as medians (inter-quartile range, IQR) and tabulated. Plasma levels of IFN-, IL-10 and

adiponectin were presented as box plots. Across-group comparisons in continuous data

were performed using Kruskal Wallis test followed by post-hoc Dunns corrections for

multiple comparisons. Chi-square test was performed for proportions while spearmans

rank correlation test was used to examine the associations of cytokines with CD4+ T-cell

counts, viral load and BMI. All tests were two-tailed and an -value of 5% (P<0.05) was

used for statistical inferences.

 36

CHAPTER FOUR

RESULTS

4.1 Baseline characteristics of the study participants

Baseline demographic, clinical and drug use characteristics of the study participants are

shown in Table 4.1. Age of the study participants did not differ significantly across the

study groups although HIV-1/TB co-infected ART naive patients tended to be older

compared to the healthy controls (P=0.057).

While weight of the study participants differed significantly across the groups (P=0.001),

their height was comparable (P=0.065). Untreated HIV mono-infected individuals

presented with lower weight compared to uninfected substance users (P=0.001) and

healthy controls (P=0.01). Additionally, BMI varied significantly across the study groups

(P<0.0001) with HIV-1 mono-infected ART-naive and HIV-1/TB co-infected ART-

exposed patients showing reduced BMI compared to healthy individuals (P=0.01).

CD4+ T cell analyses revealed significant difference across the study groups (P=0.011)

with HIV-1 mono-infected ART-naive individuals presenting with lower counts relative

to uninfected drug users (P=0.01). HIV-1 viral load did not differ significantly among

HIV-1 infected patients (P=0.081) although HIV-1 mono-infected ART naive individuals

had high viral load (median, 2,464; IQR, 52,258 copies/ml). Substance use information of

the study participants revealed that alcohol, cigarette, bhang, khat, cocktail, and

analgesics were the most commonly used non-injection drugs.

 37

Table 4.1. Baseline demographic, clinical and drug use characteristics

Healthy
Non-IDUs
controls
HIV- HIV- HIV-
HIV-1[- HIV-
HC, HIV-1[- 1[+]/ART[- 1[+]/ART[ 1[+]/ART[+
Characteristic ]/TB[-], 1[+]/ART[- P
n=27 ]/TB+, n=5 ]/TB[+], +]/TB[-] ]/TB[+],
n=32 ]/TB[-], n=6
n=12 n=9 n=21
Age, yrs. 26.0 (7.9) 31.3 (12.6) 27.9 (16.7) 29.8 (4.4) 35.2 (12.8) 32.7 (8.5) 29.6 (11.4) 0.057
Male/Female, n (%) 9/18 20/12 4/1 3/3 7/5 4/5 9/12
-
(33.3/66.7) (62.5/37.5) (80.0/20.0) (50.0/50.0) (58.3/41.7) (44.4/55.6) (42.9/57.1)
Height, m 1.6 (0.1) 1.7 (0.1) 1.7 (0.2) 1.7 (0.1) 1.7 (0.2) 1.7 (0.1) 1.7 (0.2) 0.065
Weight, kg 62 (18.0) 61.5 (13.0) 57.0 (16.0) 45.5 (9.5) 55.0 (12.0) 54.0 (7.5) 52.0 (10.5) 0.001
BMI, kg/m2 22.8 (5.3) 21.8 (5.7) 17.6 (3.2) 17.4 (3.1) 19.5 (3.4) 19.9 (2.5) 18.5 (3.0) <0.0001
CD4+ T cells/l 755 (463) 799 (499) 632 (489) 523 (237) 748 (531) 466 (414) 721 (551) 0.011
2,464
HIV-1 RNA, copies/ml - - 150 (0) 150 (3,277) 150 (0) 0.081
(52,258)
Substance use, n (%)
Alcohol 0 (0.0%) 13 (40.6) 2 (40.0) 2 (33.3) 3(25.0) 5 (55.6) 2 (9.5) -
Cigarette 0 (0.0%) 11 (34.4) 3 (60.0) 4 (66.7) 0 (0.0) 8 (88.9) 4 (19.0) -
Khat 0 (0.0%) 14 (43.8) 2 (40.0) 1 (16.7) 1 (8.3) 2 (22.2) 4 (19.0) -
Bhang 0 (0.0%) 1 (3.1) 0 (0.0) 3 (50.0) 0(0.0) 5 (55.6) 2 (9.5) -
Cocktail 0 (0.0%) 1 (3.1) 0 (0.0) 2 (33.3) 0 (0.0) 4 (44.4) 0 (0.0) -
Analgesics 0 (0.0%) 9 (28.1) 2 (40.0) 2 (33.3) 3 (25.0) 0 (0.0) 5 (23.8) -
Data presented are medians (interquartile range, IQR) unless indicated. HC, healthy controls. HIV-1, human immunodeficiency virus
type-1. BMI, body mass index. TB, tuberculosis. ART, antiretroviral therapy. Data analysis was conducted using chi-square tests for
proportions and Kruskal Wallis tests for continuous data. Values in bold are significant P-values
 38

4.2 Circulating interferon-gamma levels

Plasma IFN- levels differed significantly across study groups (P<0.001) and were

higher in ART-naive (P<0.001) and -exposed (P<0.001) HIV/TB co-infected, TB mono-

infected (P<0.01) and uninfected substance users (P<0.001) compared to healthy

controls. In addition, ART-naive (P<0.01) and -exposed (P<0.01) HIV-1/TB co-infected

individuals had significantly higher IFN- levels compared to ART-exposed HIV-1

mono-infected group (Figure 1).

P<0.0001
Plasma IFN- levels (pg/mL)

140 P<0.001
P<0.001
120
P<0.01 P<0.01
100
P<0.001 P<0.05
80
60
40
20
0
HC

HIV-1[-]/TB[-]

HIV-1[-]]/TB[+]

HIV-1[+]/ART[-]/TB[+]
HIV-1[+]/ART[-]/TB[-]

HIV-1[+]/ART[+]/TB[+]
HIV-1[+]/ART[+]/TB[-]

Figure 1. Plasma IFN- levels

Box plots are shown with the horizontal line indicating the median levels, the lower and upper
edge of each box indicate the 25th and 75th percentiles respectively while the lower and upper
whiskers show the 10th and 90th percentiles. Dots represent outliers. Arrowed lines designate
across-group difference while angled lines indicate between group comparisons. Kruskal Wallis
tests were performed for across group comparison followed by post-hoc Dunns corrections for
multiple comparisons. P-values on top of the lines indicate significant difference. HIV-1, human
immunodeficiency virus-1. TB, tuberculosis. ART, antiretroviral therapy. HC, healthy controls.
IFN-, interferon-gamma. Kruskal Wallis test was performed for across group comparison
followed by post-hoc Dunns corrections for multiple comparisons.
 39

4.3 Plasma interleukin-10 levels

Circulating IL-10 levels differed significantly across study participants (P<0.035) and

were higher in uninfected substance users compared to healthy individuals (P<0.05).

Plasma IL-10 levels were similar among HIV/TB co-infected, HIV mono-infected and

TB mono-infected groups (Figure 2).

Plasma IL-10 levels (pg/mL)

200
P=0.035
150
P<0.05

100

50

0
HC

HIV-1[-]/TB[-]

HIV-1[-]]/TB[+]

HIV-1[+]/ART[-]/TB[+]
HIV-1[+]/ART[-]/TB[-]

HIV-1[+]/ART[+]/TB[+]
HIV-1[+]/ART[+]/TB[-]

Figure 2. Plasma IL-10 levels

Horizontal line across each box shows the median levels, the lower and upper edge of
each box indicate the 25th and 75th percentiles respectively while the lower and upper
whiskers show the 10th and 90th percentiles. Dots represent outliers. Arrowed lines
designate across-group difference while angled lines indicate between group
comparisons. Kruskal Wallis tests were performed for across group comparison followed
by post-hoc Dunns corrections for multiple comparisons. P-values on top of the lines
indicate significant difference. HIV-1, human immunodeficiency virus-1. TB,
tuberculosis. ART, antiretroviral therapy. HC, healthy controls. IL-10, interleukin-10.
 40

4.4 Interferon-gamma to interleukin-10 ratios

Overall comparison revealed a strong statistical difference in IFN-/IL-10 ratios across

the groups (P<0.0001). Subsequent post-hoc comparisons showed significantly higher

IFN-/IL-10 ratio in ART-naive (P<0.001) and -exposed (P<0.05) HIV/TB co-infected

as well as TB mono-infected (P<0.01) patients compared to healthy controls. In addition,

HIV-1 mono-infected patients on treatment had lower ratios compared to untreated co-

infected (P<0.01) and TB mono-infected individuals (P<0.05; Figure 3).

P<0.0001
2.0 P<0.05
P<0.001
IFN- /IL-10 Ratio

1.5 P<0.01 P<0.01

P<0.05
1.0

0.5

0.0
HIV-1[+]/ART[-]/TB[-]

HIV-1[+]/ART[+]/TB[-]
HIV-1[+]/ART[-]/TB[+]

HIV-1[+]/ART[+]/TB[+]
HIV-1[-]/TB[-]

HIV-1[-]]/TB[+]
HC

Figure 3. IFN-/IL-10 ratios

The boxes represent inter-quartile range (IQR); line between the boxes represents the median,
while the whiskers indicate the 10th and 90th percentiles. Dots represent outliers. Arrowed lines
designate across-group difference while angled lines indicate between group comparisons.
Kruskal Wallis tests were performed for across group comparison followed by post-hoc Dunns
corrections for multiple comparisons. P-values on top of the lines indicate significant difference.
HIV-1, human immunodeficiency virus-1.TB, tuberculosis. ART, antiretroviral therapy. HC,
healthy controls. IL-10, interleukin-10. IFN-, interferon-gamma.
 41

4.5 Plasma adiponectin levels

Plasma adiponectin levels differed significantly across the study groups (P=0.036) with

HIV-1 mono-infected ART-naive patients showing lower levels compared to HIV/TB co-

infected ART-naive patients (P<0.05), un-infected non-IDUs (P<0.05) and healthy

controls (P<0.05; Figure 4).

Plasma Acrp30 levels (ng/mL)

50 P=0.036
P<0.05
40
P<0.05
P<0.05
30

20

10

0
HIV-1[-]]/TB[+]
HIV-1[-]/TB[-]

HIV-1[+]/ART[-]/TB[+]
HIV-1[+]/ART[-]/TB[-]

HIV-1[+]/ART[+]/TB[+]
HIV-1[+]/ART[+]/TB[-]
HC

Figure 4: Plasma adiponectin levels

Box plots are shown with the horizontal line indicating the median levels, the lower and upper
edge of each box indicate the 25th and 75th percentiles while the lower and upper whiskers show
the 10th and 90th percentiles. Dots represent outliers. Arrowed lines designate across-group
difference while angled lines indicate between group comparisons. Kruskal Wallis tests were
performed for across group comparison followed by post-hoc Dunns corrections for multiple
comparisons. P-values on top of the lines indicate significant difference. HIV-1, human
immunodeficiency virus-1.TB, tuberculosis. ART, antiretroviral therapy. HC, healthy controls.
Acrp30, adiponectin.
 42

4.6Correlationsofplasma cytokines with HIV-1 disease markers

4.6.1 Correlations in HIV-1 infected patients

In this study IL-10 was inversely correlated with IFN-/IL-10 ratio in ART-exposed (=-

0.959; P=0.0001; Table 4.2) and naive (=-0.932; P=0.0001; Table 4.4) HIV and TB

co-infected as well as HIV-1 mono-infected ART-exposed patients (=-0.933; P=0.0001;

Table 4.3). In addition, among HIV-1 mono-infected ART-naive patients, IFN-

(=0.943; P=0.005) and BMI (=0.829; P=0.042) were positively associated with IFN-

/IL-10 ratio while adiponectin correlated positively with the viral load (=0.829;

P=0.042; Table 4.5).

 43

Table 4.2. Correlations of cytokines with CD4+ T cell counts, viral loads and BMI in HIV-1 and TB co-infected ART-exposed
patients

IFN-/IL-10 CD4+ T HIV-1 RNA,

Marker IFN- IL-10 Adiponectin BMI
ratio cells/l copies/ml
P P P P P P P
IFN- 1.000 -
IL-10 0.201 0.394 1.000 -
IFN-/IL-10 ratio 0.024 0.920 -0.959 0.0001 1.000 -
Adiponectin 0.116 0.707 0.132 0.668 -0.203 0.505 1.000 -
CD4+ T cells/l -0.407 0.075 0.039 0.870 -0.119 0.618 -0.345 0.227 1.000 -
HIV-1 RNA,
0.037 0.879 -0.325 0.174 0.354 0.138 0.140 0.649 -0.314 0.177 1.000 -
copies/ml
BMI -0.013 0.956 0.254 0.280 -0.260 0.268 -0.020 0.946 0.236 0.303 -0.421 0.065 1.000 -

Data presented are Spearman rank correlation coefficients (rho, ) and associated P-values with significant correlation shown in bold.
IFN-, interferon-gamma. IL-10, interleukin-10. HIV-1, human immunodeficiency virus type-1. BMI, basal metabolic
 44

Table 4.3. Correlations of cytokines with CD4+ T cell counts, viral loads and BMI in HIV-1 mono-infected ART-exposed
patients

IFN-/IL-10 CD4+ T HIV-1 RNA,

Marker IFN- IL-10 Adiponectin BMI
ratio cells/l copies/ml
P P P P P P P
IFN- 1.000 -
IL-10 0.033 0.932 1.000 -
IFN-/IL-10 ratio 0.200 0.606 -0.933 0.0001 1.000 -
Adiponectin -0.433 0.244 -0.079 0.770 -0.600 0.088 1.000 -
CD4+ T cells/l -0.017 0.966 0.567 0.112 -0.600 0.088 -0.245 0.558 1.000 -
HIV-1 RNA,
-0.300 0.470 0.000 1.000 0.000 1.000 0.627 0.096 0.245 0.558 1.000 -
copies/ml
BMI -0.083 0.831 0.333 0.381 -0.367 0.332 0.367 0.332 0.233 0.546 0.464 0.247 1.000 -

Data presented are Spearman rank correlation coefficients (rho, ) and associated P-values with significant correlation shown in bold.
IFN-, interferon-gamma. IL-10, interleukin-10. HIV-1, human immunodeficiency virus type-1. BMI, basal metabolic index.
 45

Table 4.4. Correlations of cytokines, CD4+ T cell counts, viral loads and BMI in HIV-1 and TB co-infected ART-naive patients

IFN-/IL-10 CD4+ T HIV-1 RNA,

Marker IFN- IL-10 Adiponectin BMI
ratio cells/l copies/ml
P P P P P P P
IFN- 1.000 -
IL-10 -0.060 0.854 1.000 -
IFN-/IL-10 ratio 0.315 0.318 0.932 0.0001 1.000 -
Adiponectin 0.333 0.347 0.213 0.555 0.224 0.533 1.000 -
CD4+ T cells/l 0.333 0.291 0.249 0.436 -0.049 0.880 -0.479 0.162 1.000 -
HIV-1 RNA,
-0.054 0.875 0.284 0.398 -0.310 0.353 -0.342 0.367 0.121 0.722 1.000 -
copies/ml
BMI -0.452 0.140 0.312 0.324 -0.357 0.255 0.067 0.855 -0.182 0.572 0.081 0.813 1.000 -

Data presented are Spearman rank correlation coefficients (rho, ) and associated P-values with significant correlation shown in bold.
IFN-, interferon-gamma. IL-10, interleukin-10. HIV-1, human immunodeficiency virus type-1. BMI, basal metabolic index.
 46

Table 4.5. Correlations of cytokines, CD4+ T cell counts, viral loads and BMI in HIV mono-infected ART-naive patients

IFN-/IL-10 CD4+ T HIV-1 RNA,

Marker IFN- IL-10 Adiponectin BMI
ratio cells/l copies/ml
P P P P P P P
IFN- 1.000 -
IL-10 -0.406 0.425 1.000 -
IFN-/IL-10 ratio 0.943 0.005 -0.638 0.173 1.000 -
Adiponectin -0.543 0.266 0.029 0.957 -0.600 0.208 1.000 -
CD4+ T cells/l -0.086 0.872 0.203 0.700 -0.143 0.787 0.086 0.872 1.000 -
HIV-1 RNA,
-0.143 0.787 0.116 0.827 -0.313 0.544 0.829 0.042 0.143 0.787 1.000 -
copies/ml
BMI 0.771 0.072 -0.551 0.257 -0.829 0.042 -0.429 0.397 0.029 0.957 -0.029 0.957 1.000 -

Data presented are Spearman rank correlation coefficients (rho, ) and associated P-values with significant correlation shown in bold.
IFN-, interferon-gamma.IL-10, interleukin-10. HIV-1, human immunodeficiency virus type-1. BMI, basal metabolic index.
 47

4.6.2 Correlations in HIV-1 negative individuals

In TB mono-infected group, IFN- was strongly positively associated with CD4+ T cell

counts (=0.900; P=0.037) and IL-10 (=0.900; P=0.037) but negatively with IFN-/IL-

10 ratio (=-0.900; P=0.037). Additionally, there was significant inverse correlation

between IL-10 and IFN-/IL-10 ratio (=-1.000; P=0.0001; Table 4.6). However, among

HIV-negative substance users, IFN- correlated negatively with adiponectin (=-0.398;

P=0.029) but positively with IL-10 (=0.506; P=0.004; Table 4.7). Consistent with HIV-

negative substance users, the healthy control group revealed positive correlation of IL-10

with IFN- (=0.877; P=0.0001). The study also established an inverse correlation

between IL-10 and BMI among the healthy control group (=- 0.407; P=0.035; Table

4.8).
 48

Table 4.6. Correlations of cytokines with CD4+ T cell counts, and BMI in TB mono-infected patients

IFN-/IL-10 CD4+ T
Marker IFN- IL-10 Adiponectin BMI
ratio cells/l
P P P P P P
IFN- 1.000 -
IL-10 0.900 0.037 1.000 -
IFN-/IL-10 ratio -0.900 0.037 -1.000 0.0001 1.000 -
Adiponectin -0.100 0.873 0.001 0.999 0.000 1.000 1.000 -
CD4+ T cells/l 0.900 0.037 0.800 0.104 -0.800 0.104 -0.200 0.747 1.000 -
BMI 0.600 0.285 0.500 0.391 -0.500 0.391 0.500 0.391 0.700 0.188 1.000 -

Data presented are Spearman rank correlation coefficients (rho, ) and associated P-values with significant correlation shown in bold.
IFN-, interferon-gamma. IL-10, interleukin-10. HIV-1, human immunodeficiency virus type-1. BMI, basal metabolic index.
 49

Table 4.7. Correlations of cytokines with CD4+ T cell counts, and BMI in HIV-1 and TB uninfected non-injection drug users

IFN-/IL-10 CD4+ T
Marker IFN- IL-10 Adiponectin BMI
ratio cells/l
P P P P P P
IFN- 1.000 -
IL-10 0.506 0.004 1.000 -
IFN-/IL-10 ratio 0.332 0.068 -0.306 0.094 1.000 -
Adiponectin -0.398 0.029 -0.354 0.055 -0.133 0.483 1.000 -
CD4+ T cells/l -0.142 0.438 0.233 0.207 -0.141 0.450 -0.138 0.466 1.000 -
BMI 0.250 0.168 0.212 0.252 -0.088 0.638 -0.359 0.052 0.252 0.165 1.000 -

Data presented are Spearman rank correlation coefficients (rho, ) and associated P-values with significant correlation shown in bold.
IFN-, interferon-gamma. IL-10, interleukin-10. HIV-1, human immunodeficiency virus type-1. BMI, basal metabolic index.
 50

Table 4.8. Correlations of cytokines with CD4+ T cell counts, and BMI in healthy controls

IFN-/IL-10 CD4+ T
Marker IFN- IL-10 Adiponectin BMI
ratio cells/l
P P P P P P
IFN- 1.000 -
IL-10 0.877 0.0001 1.000 -
IFN-/IL-10
0.009 0.965 -0.276 0.164 1.000 -
ratio
Adiponectin -0.362 0.128 -0.323 0.177 -0.233 0.336 1.000 -
CD4+ T cells/l -0.145 0.471 -0.065 0.746 -0.040 0.843 0.212 0.383 1.000 -
BMI -0.330 0.093 -0.407 0.035 0.163 0.416 0.216 0.375 0.267 0.177 1.000 -

Data presented are Spearman rank correlation coefficients (rho, ) and associated P-values with significant correlation shown in bold.
IFN-, interferon-gamma. IL-10, interleukin-10. HIV-1, human immunodeficiency virus type-1. BMI, basal metabolic index.
 51

CHAPTER FIVE

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS

5.1 Discussion

This cross-sectional experimental study determined circulating levels of IFN-, IL-10

and adiponectin and subsequently established their correlation with CD4+ T cell

counts, viral load and BMI as markers of disease progression in HIV-1 and TB mono-

and co-infected and un-infected non-IDUs from Mombasa County. The study

revealed significantly higher IFN- levels in HIV-1 and TB co-infected ART-exposed

non-IDUs compared to healthy controls indicating that HIV-1 and TB infections

induce increased IFN- production. Elevated IFN- levels in ART-experienced HIV-1

and TB co-infected patients is suggestive of persistent immune activation despite

effective ART that indicates immunologic failure in the patients (da Silva et al.,

2013). This finding is consistent with previous studies that have reported higher

plasma IFN- levels in HIV-1 and TB co-infected patients after ART initiation

(Haddow et al., 2011).

Plasma IFN- levels were also elevated in HIV-1 and TB co-infected ART- exposed

compared to HIV-1 mono-infected ART exposed group, possibly influenced by

mycobacterial infection. Like previous studies, HIV-1 and TB co-infection increased

peripheral levels of IFN- compared to HIV-1 mono-infection (Benjamin et al.,

2013). In vitro studies have shown that stimulation of PBMCs with mycobacterial

antigens increased expression and release of IFN- in HIV-1 and TB co-infected

patients with TB-IRIS compared to non-IRIS patients (Tadokera et al., 2011; Vignesh

et al., 2013). Therefore, these results suggest that co-infection with TB in HIV-
 52

infected patients may induce overproduction of IFN-, which is associated with

profound pathological conditions in co-infected individuals.

This study also established that HIV-1 and TB co-infected ART-naive patients had

higher IFN- levels in comparison with HIV-1 mono-infected patients on ART,

indicating influence of TB as well as suppressive effect of ART on IFN- production.

This finding conforms with previous studies that have reported reduced peripheral

IFN- upon initiation of ART in HIV-1 patients (da Silva et al., 2013; Kassa et al.,

2013). Efficacy of ART has earlier been determined based on the ability of the drug to

reduce immune activation and production of inflammatory cytokines such as IFN-

and IL-10 (da Silva et al., 2013). Hence, these findings point out to the role of ART in

halting inflammation that is associated with progressive HIV disease.

Elevated circulating IFN- levels were also noted in HIV-1 and TB co-infected ART-

naive non-IDUs compared to healthy individuals, suggestive of the host immune

response to underlying disease. This is consistent with previous studies that indicate

the utility of IFN- as a key cytokine in control of TB infection (Benjamin et al.,

2013; Ashenafi et al., 2014 ).

Plasma IFN- levels were also increased in TB mono-infected individuals compared

to healthy controls, indicating that TB induces production of IFN-. This corroborates

the work of other investigators (Benjamin et al., 2013). As a protective cytokine

against TB, elevated IFN- levels in TB infected patients may indicate the hosts

adaptive mechanism used to clear the bacteria (Afum-Adjei et al., 2014), or as a

marker of TB disease severity (Adewole et al., 2013).

 53

Besides, higher plasma IFN- levels in HIV-1 and TB uninfected non-IDUs compared

to healthy controls is suggestive of substance induced inflammation. This result

coincides with the work by Neupane et al (2014) on the influence of alcohol

consumption on IFN- production. However, other studies involving cigarette (van

Zyl-Smit et al., 2014), bhang (Weiss et al., 2006) and aspirin (Medeiros et al., 2013)

have reported suppressed expression and production of IFN-. This inconsistency may

result from differences in the study designs and samples. Therefore, further research

should be carried out to ascertain these discrepancies.

Un-infected non-IDUs also had elevated levels plasma IL-10 levels compared to

healthy controls showing immuno-modulatory effect drugs and substances. Several

previous studies have shown conflicting results on the effect of drugs on IL-10

production (Neupane et al., 2014; van Zyl-Smit et al., 2014). Nicotine, an active

component of cigarette smoke for example, is reported to inhibit IL-10 expression and

production in human endothelial cells (Allam et al., 2013) and PBMCs (van Zyl-Smit

et al., 2014). In addition, in vivo studies revealed lower serum IL-10 levels in smokers

compared to non-smokers (Feng et al., 2011). On the other hand, cannabinoids, active

compounds found in bhang are reported to stimulate IL-10 production and shift Th1

to Th2 responses in mice (Weiss et al., 2006; Weiss et al., 2008). In addition, both

murine and human studies have shown that alcohol consumption stimulates release of

IL-10 in a dose-response manner (Gonzalez-Reimers et al., 2012). It is suggested that

IL-10 is produced by kupffer cells as compensatory response to counter the pro-

inflammatory activities of TNF, which drives liver inflammation in alcoholic liver

disease (Miller et al., 2011). Analgesics could also contribute to the observed raised

IL-10 levels in non-IDUs. Similarly, aspirin, a non-steroidal anti-inflammatory drug

 54

has been associated with induction of immuno-regulatory cytokines such as IL-10

(Medeiros et al., 2013), while suppressing pro-inflammatory cytokines (Berk et al.,

2013). In addition, khat, which has been commonly used by the non-IDUs, has

previously been shown to induce increased IL-10 in PBMCs (Murdoch et al., 2011).

Therefore, consumption of alcohol, bhang, aspirin and khat may induce IL-10

production, which can cause immuno-modulatory effects.

The higher IFN-/IL-10 ratios in TB and HIV-1/TB infected groups compared to

HIV-1 mono-infected individuals and healthy controls indicative of imbalance

between circulating IFN- and IL-10 levels. The IFN-/IL-10 ratio is an important

indicator of the extent of Th1 or Th2 response in HIV-1 and TB co-infection

(Benjamin et al., 2013). The higher IFN-/IL-10in TB infected patients may indicate

dominance of IFN- response while lower levels in HIV-1 mono-infected ART-naive

group suggest elevated IL-10. The ratios between pro-inflammatory and anti-

inflammatory cytokines have previously been analyzed in HIV-1 and TB co-infection

(Skolimowska et al., 2012; Benjamin et al., 2013; Mihret et al., 2014). In a recent

study, it was established that IFN-/IL-10 ratio was lower in HIV-1and TB co-

infected ART-naive patients compared to TB mono-infected patients (Benjamin et al.,

2013). However, in this study, HIV-1 and TB co-infected individuals maintained

levels of IFN-/IL-10 almost equivalent to those of TB mono-infected patients,

suggesting that TB induces higher IFN- production.

Elevated plasma adiponectin levels in HIV and TB co-infected ART-naive individuals

relative to HIV mono-infected group may signify effect of TB co-infection on adipose

tissue function as a major site of adiponectin production. However, because of the

 55

cross-sectional design of this study, it is difficult to explain the effect of elevated

plasma adiponectin levels in HIV and TB co-infected patients. Therefore, there is

need to carryout longitudinal studies to explore the role of adiponectin in HIV and TB

disease pathogenesis. Altered adiponectin levels in TB and non-TB pulmonary

diseases have been reviewed previously (Fantuzzi, 2013). Studies in chronic

obstructive pulmonary diseases (COPD) showed elevated levels of adiponectin in

patients with COPD compared to healthy individuals (Fantuzzi, 2013). In addition,

adiponectin has been suggested to be used as a marker for pulmonary TB disease

progression with patients with severe active pulmonary TB showing higher levels of

adiponectin compared to individuals with mild TB disease (Keicho et al., 2012).

Furthermore, another study also revealed elevated plasma adiponectin in active

pulmonary TB patients in comparison with household contacts and healthy controls

(Santucci et al., 2011).

The lower adiponectin levels in HIV-1 mono-infected ART-naive patients relative to

healthy controls may signify adverse effect of drug use and HIV infection on

adiponectin production. This is consistent with previous studies indicating that HIV

(Fiorenza et al., 2011) along with some drugs such as cigarettes (Tsai et al., 2011) and

alcohol (Tian et al., 2014) suppress release of adiponectin from adipose cells. It has

been established that HIV suppresses adiponectin release from adipocytes even before

initiation of treatment (Paruthi et al., 2013). Studies have shown that nicotine

stimulates oxidative stress and production of pro-inflammatory cytokine TNF-,

which inhibits expression of adiponectin gene in adipocytes (Kotani et al., 2012).

Taken together, these findings suggest that HIV-1 infection and consumption of drugs

such as alcohol and cigarette inhibit adiponectin production.

 56

In addition, decreased adiponectin levels in HIV-1 mono-infected treatment-naive

patients compared to uninfected drug users, confirms that HIV-1 infection, in partly,

suppresses production of circulating adiponectin production. This result is similar to

previous studies showing reduced plasma adiponectin levels in ART-naive and

experienced HIV-1 patients relative to their uninfected counterparts (Mody et al.,

2014; Ketlogetswe et al., 2014). While the underlying mechanisms were not

examined in the present study, it is likely that HIV infection of adipocytes causes

suppression of circulating adiponectin production. This assertion is consistent with

previous in vitro studies demonstrating that HIV-1 infection of adipocytes inhibits

adiponectin secretion by suppressing transcript and protein expression (Fiorenza et

al., 2011).

Conversely, a strong positive correlation of plasma adiponectin with HIV-1 viral load

suggests a possible link between adiponectin and HIV progression. Association of

adiponectin with viral replication has previously been established (Chiang et al.,

2013). A recent study in mice demonstrated that hepatitis B virus replication

significantly induced the increase in circulating adiponectin levels, possibly, through

activation of PPAR-gamma gene expression (Chiang, 2014). In addition, Aram et al.

(2012) established higher serum adiponectin levels in patients with persistent HIV-1

viral replication compared to patients with undetectable viral load, although

correlation of adiponectin with HIV-1 viral load was not significant. The role of

adiponectin in untreated HIV infection seems conflicting, therefore, longitudinal

studies with well-controlled models would be able to explain the arising

discrepancies.
 57

The IFN-/IL-10 ratios in this study were positively correlated with BMI in HIV-1

mono-infected ART-naive patients indicating that higher plasma IFN- levels are

associated with increase in body mass while elevated plasma IL-10 is linked to

decreased body mass. Among HIV uninfected individuals, increased BMI has

previously been associated with profound inflammation characterized by increased

circulating TNF- and IL-6 and decreased plasma IL-10 levels (Chang et al., 2013).

Similarly, in HIV-1 infection other markers of systemic inflammation such as highly

sensitive C reactive protein have been positively correlated with BMI (Ssinabulya et

al., 2014). Therefore, plasma IFN- and IL-10 can be valuable surrogate markers of

nutritional level in HIV-1 infected patients.

The study also revealed an inverse relationship between adiponectin and IFN- in

uninfected non-IDUs, suggesting anti-inflammatory activity of adiponectin. Earlier

studies established that adiponectin inhibits proliferation and cytokine production of

antigen-activated T-cells (Wilk et al., 2011). In addition, a recent experimental model

reported increased IFN- gene expression and protein levels in culture supernatants of

adiponectin knock-out mice compared with wild-type mice (Piccio et al., 2013).

Therefore, adiponectin may down-regulate drug-induced inflammation by suppressing

IFN- production.

On the other hand, among the TB mono-infected group, IFN- correlated positively

with CD4+ T cell count. Previous investigators have established that CD4+ T

lymphocytes are the primary source of IFN- during MTB infection (Green et al.,

2013), although other cells including CD8+ T cells and NK cells also produce this

cytokine (Bold and Ernst, 2012). In a recent study, it was revealed that both CD4+ T
 58

cells and IFN- produced from this T lymphocyte sub-type were essential for the

control of MTB infection and host survival (Green et al., 2013). Like the current

study, Kassa et al. (2013) established a strong positive correlation between recovery

of CD4+ T cells and IFN- secretion in active TB patients after six months of TB

treatment. Therefore, this positive correlation between CD4+ T cells and IFN- may

explain the higher TB prevalence among immune-suppressed HIV-1infected patients.

The study also established marked positive correlation of IFN- with IL-10 in TB

mono-infected, un-infected drug users and healthy controls indicating contribution of

both Th1 and Th2 cells. Previously, positive association of IFN- with IL-10 in TB

patients has been linked to increased disease severity (Skolimowska et al., 2012). As

an anti-inflammatory cytokine, IL-10 may be produced as an adaptive measure to

suppress pro-inflammatory activities to prevent damage to the host (Skolimowska et

al., 2012). However, by halting macrophage and dendritic cell functions required for

the control of initial MTB multiplication, elevated IL-10 may also be coupled to MTB

survival strategy (Skolimowska et al., 2012). Therefore, during TB infection elevated

levels of both pro-inflammatory IFN- and anti-inflammatory IL-10 may depict

impaired immune response that may lead to enhanced mycobacterial pathogenesis.

Positive correlation of IFN- with IL-10 in HIV-negative drug users may reflect

activation of both Th1 and Th2 cells induced by addictive substances (Neupane et al.,

2014). Prior studies have shown elevated IFN- and IL-10 in alcoholic cirrhotic

patients relative to non-alcoholic patients (Gonzalez-Reimers et al., 2012). In

addition, it has been demonstrated that high frequency alcohol consumption is

associated with higher circulating levels of IFN- and IL-10 (Neupane et al., 2014).
 59

Although earlier studies had shown stimulation of IFN- and IL-10 production in

cannabinoid-exposed mice (McKallip et al., 2005), other studies revealed an inverse

relationship of IFN- with IL-10 following cannabinoid exposure (Weiss et al., 2006).

Moreover, a recent study demonstrated elevated IFN- and IL-10 production by khat-

stimulated PBMCs (Murdoch et al., 2011). Therefore, elevated levels of both pro-

inflammatory IFN- and anti-inflammatory IL-10 reflect impaired IFN-/IL-10

balance among substance users.

While prospective studies with larger sample size would be more useful in explaining

cytokine dysregulations observed in HIV and TB co-infected non-IDUs, this cross-

sectional study provides the first baseline information regarding cytokine levels in

HIV-1 and TB co-infected substance users in association with disease outcome in

Kenya. In addition, a case-control study involving both HIV and TB co-infected drug

users and non-drug users can provide valuable data on the effect of substance use on

HIV and TB co-infection. Toxicological analyses can give reliable information on the

type of drugs consumed instead of self-reported information used in this study.

Furthermore, the use of plasma samples in determination of cytokine levels may not

reflect specific T cell responses during HIV and TB co-infection; therefore, in vitro

studies to determine both HIV and TB antigen-induced cytokine release and gene

expression would help understand better HIV and TB immuno-pathogenesis.

5.2 Conclusions

In summary, this study revealed elevated plasma IFN- levels in TB mono-infected as

well as in ART-nave and exposed HIV and TB co-infected patients compared to

healthy controls. In addition, both ART-nave and -exposed HIV and TB co-infected
 60

patients had higher plasma IFN- levels compared to HIV-1 mono-infected ART-

exposed patients, suggesting that TB induces increased IFN- production in these

patients. In contrast, plasma IL-10 assays revealed comparable results among HIV

and TB mono-and co-infected groups and healthy controls indicating that neither HIV

nor TB infection impairs circulating IL-10 levels in the patients. In contrast, HIV-1

and TB uninfected non-IDUs showed elevated plasma IL-10 levels indicating that

non-injection drug use may induce increased IL-10 production. There were lower

circulating adiponectin levels in HIV-1 mono-infected ART-nave compared to HIV

and TB co-infected ART-nave patients, HIV and TB un-infected non-IDUs and

healthy controls depicting detrimental effect of HIV-1 infection on adiponectin

production.

Correlation analyses established a positive association between plasma IFN- levels

and CD4+ T cell counts among TB mono-infected patients indicating that CD4+ T

cells produce IFN- during early TB. IFN-/IL-10 ratios correlated positively with

BMI in HIV-1 mono-infected ART-naive patients signifying the importance of IFN-

and IL-10 as indicators of nutritional level in HIV-1 infected patients. This is the first

study to report a significant positive correlation between plasma adiponectin levels

and viral load, therefore, further research on the role of adiponectin in HIV-1 disease

progression is needed.

These findings therefore rejected the null hypotheses that circulating IFN-, IL-10 and

adiponectin levels are similar among HIV and TB co-infected and uninfected

substance users and that the cytokines levels are not associated with HIV clinical
 61

outcomes in this population. This indicates that substance use interferes with

production of these cytokines that may influence HIV and TB disease progression.

5.3 Recommendations

This study suggests plasma IFN- can be used as a marker of early TB

infection among substance users in addition to smear microscopy, the most

commonly used TB diagnostic technique in Kenya.

Drug and substance use screening should be adopted in health care facilities as

a routine check-up among HIV-1and TB infected patients and the affected

patients should be advised accordingly.

Furthermore, evaluation of IFN-/IL-10 balance as an indicator of loss of body

mass will be important among HIV-1 patients.

5.4 Suggestions for future research

In vitro studies should be carried out to explore on the sources of IFN- during

HIV and TB infections.

Longitudinal studies should be carried out to explore the role of adiponectin in

HIV and TB pathogenesis.

 62

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APPENDICES

Appendix I: Approval letter

 72

APPENDIX II: PARTICIPANT CONSENT FORM

I am Ms. Saraphine Nebere a student at Kenyatta University. We are conducting a

study to determine cytokine levels in HIV/TB co-infected patients so that we may
optimize tuberculosis and HIV co-treatment in Africa and improve the capacity to
conduct clinical trials. The Ethical Review Committee of Kenyatta University has
approved this study.
Research Procedures
If you agree to participate in this study, you will be asked questions regarding
yourself, your health and where you reside. A registered medical laboratory
technician will take 10 millilitres of blood from you for laboratory analysis. During
the procedure, sterile and disposable instruments that are safe will be used. The blood
taken from you will be analyzed here at Bomu Medical Centre to determine your
blood count and if necessary to confirm your HIV status. The rest of the blood will be
transported to Kenyatta University for analysis of cytokine levels.
In order to ensure complete confidentiality of the test results, your names will not be
attached to the blood samples, but a number assigned to you will be used to label the
samples.
Risk/benefits
During this procedure, there will be no long-lasting effect. However, you may feel a
brief moment of pain or fear. Your will not be given any monetary benefits, and
neither will you incur any costs. The study will benefit you since we will not only
inform you of the outcomes of the assays and use them to manage you better, but we
will also be able to recommend and design appropriate interventions to minimize the
impact of these infectious diseases.
Your Rights
Your participation in this study is voluntary and if you stop participation, you will not
be denied any services that are normally available to you.
Confidentiality
We will make every effort to protect your identity. You will not be identified in any
report or publication of this study or its results.
Contact Information
If you have questions now or in the future regarding your rights or this study, you
may contact
-------
---------
---------
Consent for the individual for blood sample
May I now ask, do you agree to participate in the study?
The above details about the study and the basis of participation have been explained
to me and I agree to take part in the study. I understand that I am free to be part of the
study. I also understand that if I do not want to go on with the study, I can withdraw at
any time. I give my consent for my blood to be taken for Full Blood Count, HIV
status, CD4, Viral load and cytokines levels determination.
Please sign here or put your right hand thumb mark if you agree:
Signature/ Thumb mark---------------------------------------Date --------------------
I have adequately explained to the interviewee all issues touching on this study and
s/he has consented to participate in the study.
SignatureDate:
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APPENDIX III: QUESTIONNAIRE FOR DATA COLLECTION

Code of patient with initials:____________________________________________

District of residence:____________________________________________
Location of residence:_______________________________________
Village of residence:_____________________________________
Mobile number:__________________________________
Age:_______________________________________________________________
Sex:______________________________________________________________
HIV
Status:_____________________________________________________________
On
ART:______________________________________________________________
Which
ART:_____________________________________________________________
Duration on ART:______________________________________________________
TB status:________________________________________________________
On TB treatment:_____________________________________________________
Which anti-TB therapy:________________________________________________
Substance and drug use:________________________________________
Which drug (s):______________________________________________________
Duration of drug use________________________________________
Laboratory findings:
Body temperature (oC):____________________________________________
Height (m):____________________________________________
Weight (Kg):____________________________________________
BMI (Kg/M2):____________________________________________
Date:____________________________________________
Full Blood Count:_________________________________________
Date:_________________________________________
CD4 Count:____________________________________
Date:_________________________________________
Viral Load:____________________________________
Date:_________________________________________
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APPENDIX IV: MAP OF MOMBASA TOWN