Running Head: TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL 1

FINCTION

Type 2 Diabetes and its Impact on Neutrophil Function

Alyaa Ali Abbas

Hager Nabil Abdul Aati

Maheen Abdeulrahim Yusuf

Maryam Abdulla Yusuf

Maryam Rashed Alowain

SBS 320

December 2018

College of Health Sciences, University of Bahrain

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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
Informed Consent Form

University of Bahrain

College of Health Sciences

Medical Laboratory Program

Project Title:

Type 2 Diabetes and its Impact on Neutrophil Function

Invitation:

This project is being carried out by a group of students from College of Health Sciences,

Alyaa Ali, Maryam Rashed, Maheen Abdelrahim, Hagar Nabil and Mariam Abdullah

supervised by Dr. Mohammed Yousef.

Purpose of Study:

Before you decide to participate in this study it is important that you understand why the

research is being done and what it will involve. Please read the following information

carefully. Please ask the researcher if there is anything that is not clear or if you need more

information.

The purpose of this study is to investigate the changes in neutrophil function among type 2

diabetic patients.

Study Procedure:

In this study, a 5ml blood sample will be collected from each participant for laboratory

examination.

Benefits and Risks:

There will be no financial imputation to you for your participation in this study. However, we

hope that the information obtained from this study may help you to increase the awareness of
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
the complications that you may face as a diabetic patient and how to avoid it. On the other

hand, there are no risks for your participation in this study.

Voluntary Participation:

Your participation in this study is voluntary. It is up to you to decide whether or not to take

part in this study. If you decide to take part in this study, you will be asked to sign this

consent form.

Confidentially:

Note that your personal information collected for this study will be anonymous. Participant

data will be kept confidential.

Note: If you have any questions you are free to ask anything regarding this study at any stage.

CONSENT

I have read and I understand the provided information and have had the opportunity to ask

questions. I understand that my participation is voluntary. I understand that I will be given a

copy of this consent form.

Participant's signature ______________________________ Date __________

Investigator's signature _____________________________ Date __________

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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
Diabetes mellitus (DM), commonly referred to as diabetes, is a serious, metabolic,

chronic disease characterized by high blood glucose levels. Diabetes occurs mainly either due

to deficiency of insulin production (a hormone produced in the pancreas gland and it is

primary function is to control blood glucose levels, which is achieved by transferring glucose

from the bloodstream to the body cells where it is converted to energy) or when there is an

impairment in the utilization of insulin by the body cells (WHO 2017). As a result, glucose

starts to build up in the blood vessels leading to hyperglycemia, which in turn may cause a

destruction of small blood vessels in the kidney, eye, heart or nerves.

There are three main types of diabetes, Type1 (Autoimmune), Type2, and gestational

diabetes. Type 1 diabetes is also known as juvenile diabetes, early onset diabetes, or insulin-

dependent diabetes and characterized by a lack of insulin. This type accounts for 10% of all

diabetes cases and mainly found among adolescents.

Type 2 diabetes is the most common form of diabetes and was previously called an adult-

onset or non-insulin dependent diabetes. However, this type of diabetes arising either when

body cells are unable to utilize existing insulin properly or when insulin is produced

insufficiently. It usually develops in adults aged over 45 years, but can also occur in other age

groups such as children, adolescents, and young adults. Nearly 90-95% of all diabetes cases

worldwide are of this type.

Gestational diabetes is another form of diabetes that occurs during pregnancy and

characterized by high blood glucose above the normal range and below the diagnostic value

of diabetes. This group is at high risk to develop type 2 diabetes (WHO 2017).

Several complications are associated with type 2 diabetes involves short-term complications

and long-term complications. Hypoglycemia and hyperosmolar hyperglycemic nonketotic

syndrome (HHNS) are the major short-term complications faced by diabetic patients. On the

other hand, long-term complications are subdivided into microvascular and macrovascular
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
complications. As mentioned before, damage that occurs in tiny blood vessels

(microvascular) of kidneys, eyes, or nerves due to hyperglycemia result in severe

consequences such as nephropathy, retinopathy, and lower limb amputation. In contrast,

macrovascular complications are a heart attack, stroke, and peripheral vascular disease. In

addition, recurrent infection is another significant problem common among diabetics.

Weintrob, A.C., and Sexton, D.J. (2009) reported/noted that “diabetics receive treatment for

infections more often than non-diabetics”. Recently, several epidemiological studies have

shown that type 2 diabetics may experience changes in neutrophils function, which

contributes to increased susceptibility and severity of infections. Neutrophils are the most

abundant type of white blood cells (~50-70%) in humans and play an essential role in the

body. It is considered as the first-line defense that migrates to the site of the infection to

ingest the microorganisms which is achieved by releasing a set of enzymes which in turn

cause the killing of these organisms (Phagocytosis).

Neutrophils play an essential role to protect the host from infectious agents and it is

important cells because their ability to migrate to varies areas in the body. When the foreign

antigens (such as bacteria) entering to the body tissue, it is stimulates the chemotaxis process

which results in the release of chemokines that attract circulating neutrophils to the

endothelium. Then, several receptors expressed on the endothelial surface (E-selectin) to bind

with neutrophil receptors (L-selectin) to allow rolling of neutrophil over the endothelial

surface. After rolling, neutrophil activates their integrin receptors allowing them to adhere to

endothelial I-CAM receptors which initiate the migration of neutrophil from circulation into

the affected tissue (diapedesis) with the help of PECAM-1 receptor (a receptor present on the

endothelial surface). After neutrophil enter the tissue, the most important neutrophil function

will begin by killing the organisms through the phagocytosis process. First of all, PRRs

receptor (pattern recognition receptors) which is present on neutrophils surface will

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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
specifically recognize PAMPs receptor (pathogen-associated molecular pattern) which found

on many microorganisms. Then, the plasma membrane of the neutrophil extended around the

foreign body and start to engulf it. After that, the engulfed particles are surrounded by a

membrane-bound vacuole called phagosome followed by fusion of lysozyme with

phagosome to form a phagolysosome. As a result of this fusion, the lysozyme will release

hydrolytic enzymes which caused an acidic environment in the sac that lead to a destruction

of the microbes. Finally, the cellular waste products will be discharged from the cell through

exocytosis process. There are two mechanisms neutrophils used in order to destroy the

microbes after engulfment either by used oxygen-dependent mechanism that depends on the

production of reactive oxygen species (ROS) or oxygen-independent mechanism.

Oxygen-dependent mechanism start when NADPH (One of the most significant components

of neutrophils which is scattered in plasma membrane and cytoplasm during resting state)

moves from plasma membrane to phagosome membrane and reduced to NADP and H +¿¿ by

an oxidation process. At the same time, this step will lead to oxidation of O2 into O2
−¿ ¿

(superoxide anion). After that, superoxide anion will react with H +¿¿ to produce H 2 O2

(Hydrogen peroxide) but this molecule is not very efficient as bactericidal agent, so in the

presence of MPO (myeloperoxidase enzyme that found naturally in azurophilic granules of

neutrophil) and Cl−¿¿(Chlorine) hydrogen peroxide will be converted to hypochlorite and this

product is more effective as bactericidal agent . Collectively, superoxide, hydrogen peroxide

and hypochlorite are called ROS.

The other mechanism is the oxygen-independent mechanism. This mechanism is not effective

as the oxygen-dependent ones and it has four main types. The first one uses cationic peptides

that damage the bacterium's membrane. The second type uses lysozymes that break down

peptidoglycan. The third type uses lactoferrins, which are a protein that prevents bacteria

from needed iron and present in neutrophil granules. The last type uses hydrolytic enzymes
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
and cathepsin G in which these enzymes cause damage to microbial membranes. The global

prevalence of diabetes has been steadily increasing over the past few decades from 4.7% in

1980 to 8.5% in 2014 (WHO 2017). In addition, the highest prevalence in Middle East and

North Africa region are mainly found in Saudi Arabia (18.5%), Bahrain (16.2 %), UAE

(15.6%), Egypt and Kuwait (15.1 %) (IDF 2017).

Literature Review

PubMed, Google Scholar, and MEDLINE were searched to identify studies published

between 2001 and 2013 reporting the relationship between type 2 diabetes and neutrophil

function. There was a study done by McManus, Bloodworth, Prihoda, Blodgett, and Pinckard

in 2001 with a title of (Agonist-dependent failure of neutrophil function in diabetes correlates

with extent of hyperglycemia). The purpose of this study was to determine if there is a

relationship between increasing of blood sugar and leukotriene B4 synthesis (LTB4) or

lysosomal-enzyme secretion in isolated neutrophils stimulated with different agonists.

Through the study, researchers used some agonists such as calcium ionophore (A23187),

formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) that can

modulate neutrophils activation by distinct signal transduction mechanisms. Ultimately, they

found that neutrophils dysfunction in diabetes patients occurred only with stimuli initiate

signal transduction through GPCR (agonists acting via G-protein-coupled receptors such as

fMLP and PAF) and this deficit inversely correlated with glycemic control. Thus, they

conclude that neutrophils function are progressively decreased as blood glucose levels are

increased, as well as, there were significant reductions of LTB4 production and lysosomal-

enzyme secretion.

One of the studies was carried out in 2004 by Gorudko et al. in order to investigate the

functional activity of neutrophils during type 2 DM and coronary heart disease (CHD),

particularly the role of the myeloperoxidase enzyme in the development of the oxidative
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
stress. In this study, the researchers studied peroxidase activity of myeloperoxidase, plasma

catalase activity, and levels of TBA-reactive lipid peroxidation products and oxidized

glutathione enzyme in three groups (healthy individuals, type 2 diabetic patients and type 2

diabetic patients with symptoms of CHD). Neutrophils have been stimulated to produce

H 2 O2 by using a specific concentration of the N-formyl-met-leu-phe (fMLP) which serves as

a potent polymorphonuclear leukocyte (PMN) chemotactic factor. At the end of the study, the

researchers found that there was a marked increase in myeloperoxidase activity in patients

with type 2 DM. Since myeloperoxidase is considered as a significant neutrophilic mediator

in the prevention of oxidative stress, its increased activity serve as an additional indicator of

oxidative stress in patients with DM. This, in turn, confirms the fact that patients with type 2

DM have a defect in the function of neutrophils since myeloperoxidase enzyme is found

mainly in these cells and play a key role in the performance of its function.

Impaired chemotaxis, defective phagocytosis, and increased production of free radicals are

problems have been reported in diabetes mellitus. A respiratory burst that occurs via

activating the NADPH oxidase is one of the most important steps to stimulate phagocytosis

and thus kill bacteria. (Nicotinamide Effects Oxidative Burst Activity of Neutrophils in

Patients with Poorly Controlled Type 2 Diabetes Mellitus) is another study conducted in 2004

by Osar, Samanci, Demirel, Damci, and Ilkova. In this study, researchers hypothesized that

nicotinamide may restore the impaired oxidative burst capacity of neutrophils in diabetic

patients by increasing the NADH content as an electron donor and possibly through NADPH

oxidase activity. In order to examine this hypothesis, these experts divided the diabetics into

groups where one group was given nicotinamide and the other group received placebo for one

month. Eventually, scientists found that the oxidative burst activity in patients receiving

nicotinamide was greater when compared with the placebo group and the difference was

statistically significant at 30 and 45 minutes. This demonstrates that the dysfunction of

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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
neutrophils found in diabetic patients is occurring mainly due to low NADPH level along

with the decreased activity of NADPH oxidase, which in turn are considered the key

elements used in the production of H 2 O2 and other oxidizing radical (Ex, oxidized halogens).

Impaired phagocytosis of capsular serotypes K1 or K2 Klebsiella pneumoniae in type 2

Diabetes mellitus patients with poor glycemic control was a study done by Lin et al. in 2005.

According to this study, the researchers presented that patients with liver abscess and septic

endophthalmitis due to K.pneumoniae are frequently associated with type 2 Diabetes. In

addition, 93% of endophthalmitis patients and 75% of liver abscess patients due to

K.pneumoniae were suffering from type2 DM. This study was done on three groups, in which

two groups had type 2 diabetes but one group with good glycemic control and the other group

with poor glycemic control and the third group was healthy control. The purpose of this

investigation was to estimate the phagocyte activity by compared neutrophil phagocytosis of

serotypes K1/K2 and non- K1/K2 K. pneumoniae among type 2 diabetes patients (with either

good or poor glycemic control) with healthy individuals by using Fluorescence technique

(fluorescein isothiocyanate) and electron microscopy. The result showed that the neutrophil

phagocytosis of K1 and K2 sera was low in patients with type 2 diabetes comparing to

healthy individuals. On the other hand, no different in phagocytosis was observed in non-K1

and K2 sera. Additional observation showed that diabetes patient with good glycemic control

has normal phagocytes activity. The researchers conclude that the major factor affecting

phagocytosis is diabetes and bacterial factors.

Another experimental study was done by Alba-Loureiro et al. in 2007 to examines the

relationship between biochemical metabolism and neutrophil function in rats with type 2

diabetes. The researchers studied the maximum activities of hexokinase enzyme (glycolytic

pathway) and citrate synthase enzyme (Krebs cycle) of neutrophil metabolism. At the end of

the study, they demonstrated that neutrophils from diabetic rats exhibit impairment in the
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
utilization of glutamine and glucose thus insufficient ATP synthesis and they conclude that

leukocyte dysfunction is linked to metabolic pathways.

Shetty, Thomas, and Ramesh in 2008 made a study with a title of (Comparison of Neutrophil

Functions in Diabetic and Healthy Subject with Chronic Generalized Periodontitis). The aim

of this study was to evaluate the neutrophil function in 15 diabetic patients with chronic

generalized periodontitis. In order to study the neutrophil function, the researchers evaluate

chemotaxis, superoxide production, phagocytosis and killing of Porphyromonas gingivalis

(bacteria that cause periodontitis) by diabetic neutrophils relative to healthy and matched

control. At the end of the study, they observed significant depression in numbers of diabetic

neutrophils migrating, as well as critical enhancement of diabetic neutrophil superoxide

production, and impaired phagocytosis. So the result explained that chronic generalized

periodontitis in the diabetic patient is associated with neutrophils malfunction.

An interesting study was done by Khan et al in 2013 with a title of (Impairment of

Mitochondrial-Nuclear Cross Talk in Neutrophils of Patients with Type 2 Diabetes Mellitus).

In this study, the researchers evaluate the mitochondrial oxidative stress, levels of nuclear

DNA damage and apoptosis in peripheral blood neutrophils isolated from type 2 diabetes

mellitus patient (T2DM). For participation process, the diabetic participant was divided into

two groups: early and late onset of T2DM patients, and both participants were required to

have relatively stable glycemic control, manifested by at least successive glycated

Hemoglobin (HbA1C) previous assay results with no more than 1% variation. However, they

observed an elevation of mitochondrial oxidative stress in both early and late diabetes with a

higher level of H 2 O2 in neutrophils of late T2DM patients, in addition to consistent

diminution in levels of antioxidant activity in peripheral neutrophils in T2DM patients which

eventually result in impairment of neutrophil homeostasis and function.

Prevalence
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
Globally

The global prevalence of diabetes shown that the number of people with diabetes has

risen from 108 million in 1980 to 422 million in 2014, 422M are mostly adults over 18 years

old.

The report of WHO (2016) shown that the largest population with Diabetes disease are

present in South-East Asia and Western Pacific Regions. In few decades the same report has

shown that the numbers of Diabetes are raises rapidly due to same reasons, for example,

increase the age average of the population, population growth and increase in the prevalence

of diabetes at each age.

The mortality of people with diabetes was 1.5 million in 2012, it was conceded the eighth

leading cause of death during that years. Almost most of the death cases in diabetes are due to

high blood glucose level. The great mortality case due to the high glucose level occurs in
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
upper-middle income countries (1.5 million) and the lowest number in low-income countries

(0.3million).

Although Type 2 Diabetes seen mostly in adult and elderly people, now it’s arise in young

and children. There are no data on the true incident about the type 2 patient because most of

the time it is undiagnosed. The report from seven countries found that 24%-62% of people

with diabetes are not diagnosed and not treated. (WHO 2016).

Regionally

Recently, the prevalence of diabetes has been increased significantly in the Middle East

and North Africa (MENA) region. According to the International Diabetes Federation (IDF)

in 2017, almost 38.7 million adults aged 20-79 years were found in the MENA region. Nearly

to half (49.1%) of these cases were undiagnosed. More than two-thirds (67.3%) of adults with

diabetes live in urban regions.

MENA region becomes one of the highest rates of diabetes in the world. In addition, the

prevalence of diabetes has demonstrated a growing burden, and among the three types of

diabetes, type 2 diabetes is predominating while type 1 diabetes and gestational diabetes are

less common. However, the Gulf region has the highest prevalence among the MENA region,

four of the Gulf countries (Saudi Arabia, Bahrain, Kuwait, and United Arabic Emirate)

reporting the prevalence of equal to or more than 15% while Oman and Qatar with a

prevalence of less than 15% (IDF, 2017). The reason of increasing the prevalence in the Gulf

region is their recent societal changes with an elevation of economic development and growth

associated with decreased infant mortality and increasing life expectancy, resulting in a huge

change in the lifestyles, diet and physical activity in their population. However, Saudi Arabia

ranked first in the MENA region with a prevalence of 18.5% in 2017, while Yemen ranked

last with a prevalence of 3.8% in 2017 (IDF 2017).

Country/territory Prevalence(%), 2017

Saudi Arabia 18.5
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
Kuwait 15.1
Qatar 14.1
Bahrain 16.2
United Arab Emirates 15,6
Egypt 15.1
Lebanon 14.6
Oman 10.7
Jordan 9.5
Iran 8.9
Afghanistan 6.7
Algeria 6.9
Libya 11.2
morocco 7.3
Pakistan 6.9
Palestine 7.0
Sudan 10.9
Syria 7.5
Yemen 3.8
Table1: the prevalence in the adult of the MENA region in 2017 (IDF)
Nationally

Bahrain has experienced a marked and rapid development in its socio-economic status,

leading to significant changes in lifestyle and patterns of health and disease. There has been a

sharp decline in infectious diseases and a rise in genetic diseases, including diabetes, which

has become one of the most common health problems in the kingdom.

According to the International Diabetes Federation, 5 out of 19 countries in the Middle East

and North Africa (MENA) with the highest prevalence of type 2 diabetes are the Gulf States,

including Bahrain, which ranks second where the prevalence rate reached 16.2 %. In 2016,

the number of diabetes cases among Bahrainis was 52,806 (Ministry of Health 2016). In

2017, this number has increased sharply, making the total number of diabetes cases become

165,300 (International Diabetes Federation 2017).

Bahrain has witnessed a significant increase in diabetes mortality. Diabetes mortality rate was

10% in 2012, but this percent gradually rose to become 15% by the end of 2013 (Ministry of

Health 2012,2013). The probability of premature death from one of the non-communicable
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
diseases (NCDs), including diabetes is 13%, which means nearly one out of five adults die

from NCDs before age 70 (UN Task Force on NCDs 2017).

Obesity, physical inactivity, tobacco use, and an unhealthy diet have played an important role

in increasing diabetes among Bahrainis. As a result, recent estimates suggest that diabetes

prevalence will increase by more than two-fold in Bahrain in the coming two decades,

making it a national issue that needs to be taken with high importance.

Lab procedures

One of the most important tests to detect neutrophils function is nitroblue tetrazolium test

(NBT). NBT is a qualitative test that used to measure the ROS production particularly

superoxide in neutrophil and the ability of neutrophil to perform phagocytosis. A colorless

chemical called nitro blue tetrazolium is added to white blood cells in the lab to check

whether there is change on its color or not. Normally, neutrophil changes the colorless

compound of NBT into dark blue aggregation and this indicates that cells should be able to

kill bacteria and protect the body from infections. But If there is no change in color when

NBT is added this indicates a reduced in ROS production due to NADPH oxidase defect and

thus dysfunctional neutrophils.

Another test is done in the lab in order to asset neutrophilic function is Myeloperoxidase

(MPO). MPO enzyme is one of the most abundant proteins present in the neutrophils, it is

encoded by the MPO gene on chromosome 17, stored in the azurophilic granules and released

when neutrophils are stimulated. MPO is involved in microbe clearance by neutrophils both

intracellularly (via the production of Hypochlorous acid (HOCl) ) and extracellularly (via the

release of NETs). MPO oxidizes tyrosine to tyrosyl radical using H 2 O2 as an oxidizing agent.

Hypochlorous acid and tyrosyl radical are cytotoxic so they are used by neutrophil to kill

bacteria and other pathogens.

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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
Alkaline leukocyte phosphatase test also called Neutrophil alkaline phosphatase blood test it

is a test used to measure alkaline phosphatase enzyme and tells how much of this enzyme is

presented in neutrophils. A blood sample is collected in (sodium or lithium heparin) tube then

separation of the neutrophils from the rest of the WBCs after that a blood smear is prepared

in order to stain and examine, the test involves counting 100 neutrophils (segmented and

band forms only ) then neutrophil leukocytes are scored from 0 to 4+ on the basis of the

intensity of the precipitated dye in their cytoplasm. The values of the 100 cells are added and

the total score reported.

Literature Read & literature rejected:

summary of literature review:

methodology:
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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL

References

Alba-Loureiro, T. C., Munhoz, C. D., Martins, J. O., Cerchiaro, G. A., Scavone, C., Curi, R.,

& Sannomiya, P. (2007). Neutrophil function and metabolism in individuals with

diabetes mellitus. Brazilian Journal of Medical and Biological Research, 40(8), 1037-

1044.

Gorudko, I. V., Kostevich, V. A., Sokolov, A. V., Shamova, E. V., Buko, I. V.,

Konstantinova, E. E., ... & Panasenko, O. M. (2012). Functional activity of

neutrophils in diabetes mellitus and coronary heart disease: role of myeloperoxidase

in the development of oxidative stress. Bulletin of experimental biology and

medicine, 154(1), 23-26.

IDF Middle East and North Africa. (2017). Retrieved from https://www.idf.org/our-

network/regions-members/middle-east-and-north-africa/welcome.html

Khan, S., Raghuram, G. V., Pathak, N., Jain, S. K., Chandra, D. H., & Mishra, P. K. (2014).

Impairment of mitochondrial–nuclear cross talk in neutrophils of patients with type 2

diabetes mellitus. Indian Journal of Clinical Biochemistry, 29(1), 38-44.

Lin, J. C., Siu, L. K., Fung, C. P., Tsou, H. H., Wang, J. J., Chen, C. T., ... & Chang, F. Y.

(2006). Impaired phagocytosis of capsular serotypes K1 or K2 Klebsiella pneumoniae

in type 2 diabetes mellitus patients with poor glycemic control. The Journal of

Clinical Endocrinology & Metabolism, 91(8), 3084-3087.

McManus, L. M., Bloodworth, R. C., Prihoda, T. J., Blodgett, J. L., & Pinckard, R. N. (2001).

Agonist‐dependent failure of neutrophil function in diabetes correlates with extent of

hyperglycemia. Journal of Leukocyte Biology, 70(3), 395-404.

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TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
Osar, Z., Samanci, T., Demirel, G. Y., Damci, T., & Ilkova, H. (2004). Nicotinamide effects

oxidative burst activity of neutrophils in patients with poorly controlled type 2

diabetes mellitus. Journal of Diabetes Research, 5(2), 155-162

Shetty, N., Thomas, B., & Ramesh, A. (2008). Comparison of neutrophil functions in diabetic

and healthy subjects with chronic generalized periodontitis. Journal of Indian Society

of Periodontology, 12(2), 41.

Weintrob, A. C., & Sexton, D. J. (2009). Susceptibility to infections in persons with diabetes

mellitus. Waltham (MA): Uptodate.

World Health Organization. (2016). Global report on diabetes. World Health Organization.

WHO | Diabetes mellitus. (2018). Retrieved from

http://www.who.int/mediacentre/factsheets/fs138/en/