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UNIVERSITY OF DAR ES SALAAM

DEPARTMENT OF BIOCHEMISTRY
SCHOOL OF HEALTH SCIENCES

ASSESSMENT OF THE EFFECT OF MODE OF INSULIN STORAGE ON ITS


THERAPEUTIC POTENCY AMONG PATIENTS WITH TYPE 1 DIABETES
RECEIVING CARE AT CHAMWINO DISTRICT HOSPITAL, DODOMA,
TANZANIA

INVESTIGATOR
DR. NGWEINA FRANCIS MAGITTA, MD, PHD

ABSTRACT
Type 1 diabetes (T1D) is an important childhood illness that causes significant morbidity and
mortality in developing countries. The mainstay for management of T1D is the daily
administration of insulin preparations. Insulin preparations require appropriate handling and
storage at temperatures that ensures preservation of their biological and therapeutic potency.
Typically the storage requires the use of refrigerators which are often not available in the
majority of households in the rural settings in Tanzania.
Previous reports indicate that most countries in Africa, have adapted the use of diverse
traditional methods for storage of insulin preparations. However, the efficacy of these
methods on the preservation of insulin potency has not been systematically evaluated for
validation. The proposed study aims to investigate the effect of these storage methods by
indirect assessment of patients glycaemic control based on three-point measurements of
Fasting Blood Glucose (FBG). The proposed cross-sectional descriptive study, involving
patients aged 35 years solely on insulin therapy for T1D, will be conducted within
Outpatient Department (OPD) at Chamwino District Hospital in Dodoma, Tanzania.
All eligible patients receiving care at the health facility will be recruited into the study prior
to obtaining consent from the patient or where necessary from patients parents or relatives.
Important demographic variables and clinical data will be collected from patients using
structured Clinical and Laboratory Data Sheets together with supplemental data obtained
from case notes and patients guardians and relatives. Data will be analyzed using Excel
spreadsheet software and a p-value of less than 5% will be considered statistically significant.
Ethics approval will be sought from UDSM and Chamwino District Health Authority. The
fieldwork will be conducted for one month at an estimated budget of TZS 850,000. The
findings from this study will provide evidence and opportunity for improving the
management of T1D in rural settings in Tanzania.

TABLE OF CONTENTS
ABSTRACT ................................................................................................................................................. 2
List of Abbreviations and Acronyms ....................................................................................................... 4
1.

BACKGROUND .............................................................................................................................. 5

2.

LITERATURE REVIEW ................................................................................................................ 5

3.

PROBLEM STATEMENT ............................................................................................................. 8

4.

RATIONALE .................................................................................................................................... 8

5.

GOAL AND OBJECTIVES ........................................................................................................... 8


5.1.

BROAD OBJECTIVE ........................................................................................................ 8

5.2.

SPECIFIC OBJECTIVES ................................................................................................... 8

6.

MATERIALS AND METHODS................................................................................................... 9


6.1.

STUDY AREA AND STUDY DESIGN ............................................................................ 9

6.2.

STUDY POPULATION ..................................................................................................... 9

6.2.1. INCLUSION CRITERIA: .................................................................................................... 9


6.2.2. EXCLUSION CRITERIA: ................................................................................................... 9
6.3.

SAMPLING AND SAMPLE SIZE ESTIMATION ........................................................... 9

6.4.

DATA COLLECTION ..................................................................................................... 10

7. DATA MANAGEMENT AND STATISTICAL ANALYSIS .................................................... 10


7.1. DATA MANAGEMENT........................................................................................................... 10
7.2. STATISTICAL ANALYSIS ..................................................................................................... 10

7.

ETHICAL CONSIDERATIONS ................................................................................................ 11

8.

DISSEMINATION OF RESEARCH FINDINGS.................................................................. 11

9.

LIMITATION OF THE STUDY ................................................................................................ 11

10.

PROJECT ACTIVITIES AND MILESTONES ................................................................... 11

11.

BUDGETARY ALLOCATION AND ITS JUSTIFICATION .......................................... 12

12.

REFERENCES ............................................................................................................................ 12

LIST OF ABBREVIATIONS AND ACRONYMS

AIDS

Acquired Immunodeficiency Syndrome

CDC

Chamwino District Council

CID

Covalent insulin dimmers

CIP

Covalent insulin protamine

DNA

Deoxyribonucleic acid

FBG

Fasting Blood Glucose

HIV

Human Immunodeficiency Virus

HMWT

High Molecular Weight Transformation

IDF

International Diabetes Federation

NCDs

Non-Communicable Diseases

NPH

Neutral Protamine Hegedorn

OHAs

Oral Hypoglycaemic Agents

OPD

Outpatient Department

UDSM

University of Dar es Salaam

SoHS

School of Health Sciences

TID

Type 1 diabetes

TZS

Tanzanian Shillings

WHO

World Health Organization

1. BACKGROUND
Rural areas in developing countries are frequently challenged by the existing cold-chain
delivery and maintenance systems required for preservation of potency for specific drugs,
vaccines and other biologics. In particular, insulin which is required by patients suffering
from T1D requires storage at appropriate temperatures in order to ensure preservation of their
therapeutic potency.

In rural settings the recommended storage facilities are often not

available. Thus patients struggle to store their insulin preparations in facilities that might
expose insulin to increased biodegradation and progressive loss of biological and therapeutic
efficacy.

2. LITERATURE REVIEW
The incidence of diabetes and other chronic non-communicable diseases (NCDs) is
increasing world-wide. The on-going transition in the disease pattern from infectious disease
to chronic non-communicable diseases is currently the global health focus. There is lack of
data on diabetes in countries in Africa including Tanzania. However, the available evidence
indicates that the majority of patients with diabetes typically remain in the community
undiagnosed or those diagnosed usually present late to the health facilities often with
complications or dies outside hospitals
The 7th Edition of the International Federation on Diabetes (IDF) and World Health
Organization (WHO) report indicates that a total of 415 million people are estimated to have
with diabetes worldwide, representing 1 person out of 11 living with diabetes. Further, it is
noted that every 6 seconds a person dies from diabetes representing about 5 million deaths
globally. An estimated 14.2 million adults aged 20-79 years have diabetes in the IDF Africa
region, representing a regional prevalence of 3.2% (with range of 2.1-6.7%). Notably,
diabetes contributes about 321,100 deaths in the IDF Africa region. In IDF Africa region,
T1D among children aged below 15 years is estimated to affect about 46,400 children; with
about 7,600 children being diagnosed annually. However, these figures are mere estimates
are likely to be grossly underestimated due to the fact that the mortality of children with T1D
is estimated to be significantly in Africa. Typically many children with T1D lack access to
insulin, glucose test-strips and appropriately-trained health professionals. These factors
together with other prevalent diseases contribute to the increased vulnerability of children to
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poor glycaemic control and subsequent higher mortality. The prevalence of diabetes in
Tanzania is estimated at 3.5% in a total adult population of 23,514 aged 20 79 years. The
prevalence of Type 1 diabetes in most African populations is largely unknown. However,
several studies have shown a very low glycaemic control based on HB1c measurements. For
instance, a study Muze CK et al conducted in Tanzania indicates prevalence of glycaemic
control of about 49.8% based on HbA1c < 11% criteria [1].

The mainstay for management of T1D is the daily administration of insulin preparations.
Preservation of therapeutic potency of insulin preparations requires handling and storage at
appropriate temperatures. It is well-known that many drugs, biological products, and vaccines
are temperature-sensitive. One life-saving drug, which needs to be stored under temperatureregulated condition, is insulin [2]. Insulin is a life-saving product used for the management
of all type 1, and many type 2 diabetes patients. It is a polypeptide, produced by recombinant
DNA technology, using either E. coli or Saccharomyces sp [3]. Insulin analogs or modern
insulins are also available, which are produced by changing the chemical structure of the
insulin molecule. Insulin can be classified as rapid or fasting acting, e.g., human insulin,
aspart insulin, lispro insulin, and glulisine insulin; pre-mixed e.g., pre-mixed human insulin,
aspart and lispro; intermediate- acting, e.g., neutral protamine hegedorn (NPH) or iSoHSane
insulin, and long acting, e.g., detemir and glargine insulin.

For optimal effect, insulin need to be stored under refrigerated conditions, between 2 and
8C, and be protected from light when vials or pens are unopened. Pens or vials in use may
be kept at room temperature, protected from sunlight, up to 25C. A refrigerator is the best
place to store insulin in. Insulin should never be frozen as this will make it lose its potency.
Unopened insulin is best kept at 2-8C temperature. This temperature is maintained below the
freezer or in the butter compartment of most fridges. Opened insulin may be kept at room
temperature, or in the vegetable compartment of the fridge, where temperature is maintained
at a stable 14C [4]. It is further advised to avoid keeping insulin in the door of the fridge, as
this area is most prone to temperature fluctuations. Exposure to higher temperatures during
storage and use may degrade insulin by hydrolysis, or transform it to higher molecular weight
components [5]. A study conducted in India Vimalavathini R et al showed that storage of
regular and biphasic insulin at 32C and 37C decreased the potency of insulin by 14 to 18%
[6].

Temperatures below or beyond the recommended for optimal storages leads to


biodegradation and or loss of native conformation which results to loss of biological activity
and therapeutic potency. The mechanisms for degradation of insulin are diverse depending
on the degree of storage temperatures together with the type of solvents, pH and insulin
preparations e.g. soluble insulin versus preparations containing zinc and protamine [7, 8].
Insulin deteriorates rapidly in acid solutions due to extensive deamidation at residue AsnA21.
In neutral formulations deamidation takes place at residue AsnB3 at a substantially reduced
rate under formation of a mixture of isoAsp and Asp derivatives. The rate of hydrolysis at B3
is independent of the strength of the preparation, and in most cases the species of insulin, but
varies with storage temperature and formulation [9]. In another study it was revealed that
storage of insulin preparations at 4-45C resulted in the formation of covalent, higher
molecular weight transformation (HMWT) products. The main products are covalent insulin
dimers (CID), but in protamine-containing preparations the concurrent formation of covalent
insulin-protamine (CIP) products takes place. At temperatures greater than or equal to 25C
parallel consecutive formation of covalent oligo- and polymers can also be observed. Rate of
HMWT is only slightly influenced by species of insulin but varies with composition and
formulation, and for NPH preparations, also with the strength of preparation. In this study,
temperature was shown to exert a pronounced effect on CID, CIP, and, especially, covalent
oligo- and polymer formation [7].
Typically the storage requires the use of refrigerators which are often not available in the
majority of households in the rural areas in Tanzania. Several studies conducted in Africa and
other developing countries indicate the widespread use of diverse locally adopted options for
storage of insulin preparations such as cold clay pot, bowl of water, earthenware pitcher,
thermo cool boxes with ice packs and cool wet cloth around insulin vial [10]. A study in
Zambia by Allen, S.C revealed preservation of therapeutic potency when stored in a cool
place [10]. In other recent studies, storage of insulin in the clay pot in the desert has been
shown to maintain its biological activity [11, 12]. However, in most of these studies these
local and traditional insulin storage methods have not systematically evaluated. The proposed
study takes advantage of the existence of methods to evaluate the therapeutic efficacy of
insulin preparations through indirect assessment of glycaemic control in the context of
acceptable adherence of patients to the prescribed insulin therapy.

3. PROBLEM STATEMENT
T1D is a common childhood endocrine disorder that require prompt daily administration of
potent insulin preparations to achieve adequate glycaemia. Often, the storage of insulin
preparations poses significant challenges particularly in the rural settings in the tropics. The
existing practice of storage of insulin in poorly evaluated methods may contribute to poor
glycaemic control together with morbidity and mortality associated with T1D.

4. RATIONALE
To our understanding this is the first proposed survey for insulin storage modalities in
Tanzania. This survey is therefore expected to generate evidence for various storage
modalities of insulin rural settings and provide opportunity for improvement of T1D in
Tanzania. This approach will provide opportunity for optimizing the existing traditional
insulin storage modalities in order to prevent T1D-associated morbidity and mortality in
Tanzania.

5. GOAL AND OBJECTIVES


The proposed survey seeks to assess the impact of various insulin storage modalities on
insulins biological and therapeutic potency based on glycaemic control through FBG
estimates.

5.1.

BROAD OBJECTIVE

To determine the type of insulin preparations, modalities of insulin storage and its impact on
the glycaemic control among patients receiving care at a secondary care health facility in
Tanzania.

5.2.

SPECIFIC OBJECTIVES

In order to achieve the above goal, we plan:1) To identify various types of insulin preparations prescribed for treatment of patients
with T1D receiving care at Chamwino District Hospital, Dodoma, Tanzania.
2) To determine various insulin storage modalities used by patients with T1D receiving
care at Chamwino District Hospital, Dodoma, Tanzania.
3) To assess the therapeutic efficacy of insulin preparations used for treatment of
patients with T1D in relation to glycaemic control based on FBG measurements
among patients receiving care at Chamwino District Hospital, Dodoma, Tanzania.
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6. MATERIALS AND METHODS


6.1.

STUDY AREA AND STUDY DESIGN

This proposed survey is planned to be carried out in Chamwino District Hospital in Dodoma,
Tanzania. The district is located in the central plateau of Tanzania which extends between
Latitude 40 and 80 South and between longitude 35and 37 East. The district is dissected
from Northwest to Southwest by a number of mountain chains, between and around these
mountains and hill ranges there are lower-lying relatively flat areas of about 1200m elevation
[13]. The district has a total area of 8,056 square kilometers with five divisions, thirty two
wards, and seventy eight villages. The district has a dry savannah climate characterized by a
long dry season (April to early December) and a short single wet season (December to April).
The average annual rainfall is 500mm typically raining between December and March. The
major economic activities are cattle rearing and crop husbandry [13].

The proposed study is a cross-sectional descriptive study. In this study the investigator will
recruit eligible patients in the OPD receiving insulin therapy for T1D. In these patients the
investigator will in parallel document the type of insulin preparation received and storage
modality as well as the assessment of the glycaemia based on FBG levels.

6.2.

STUDY POPULATION

The proposed study will recruit patients at OPD receiving insulin therapy for T1D. The
patients with T1D will be seen and interviewed once during the course of this survey.
6.2.1. INCLUSION CRITERIA: All patients, aged 35 years diagnosed with T1D
exclusively receiving insulin therapy for over 3 months.
6.2.2. EXCLUSION CRITERIA: Patients with T1D receiving insulin therapy together with
Oral Hypoglycaemic Agents (OHAs) or those with other severe co-morbid conditions e.g.
advanced stage of HIV/AIDS, tuberculosis and obviously diabetic complications.

6.3.

SAMPLING AND SAMPLE SIZE ESTIMATION

We plan to recruit patients with T1D previously diagnosed based on the hospitals existing
criteria fulfilling the inclusion criteria. The estimation of sample size will be based on the
previously reported prevalence of glycaemic control among patients with T1D of 49.8%
according to Muze CK et al [1]. In this survey the sample size is calculated using the formula;

Where:
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n = minimum sample size required,


p = incidence of T1D (estimated at 48.9% according to a previous study)
= margin of error; set at 5% (0.05) and
z = z-score (1.96 at 95% confidence level)
From the above formula, the estimated sample size is 785. However, due to the limitation of
the allowed period for data collection it may be prudent to revert to a convenient sample of
study participants over the course of 2 months.

6.4.

DATA COLLECTION

Basic demographic variables e.g. patient ID, age and sex and contact will be collected and
documented in routine manner using a standard clinical sheet. Other patient variables related
to diagnosis, treatment, insulin storage modalities together with glycaemic control will be
collected using standard questionnaire. Finger prick blood collection will be performed in a
standard manner in the morning after an overnight fasting for assessment of FBG using the
available rapid glucose meters. In parallel, routine case notes and hospital records will be
provide supplemental data related to glycaemic control.

7. DATA MANAGEMENT AND STATISTICAL ANALYSIS


7.1. DATA MANAGEMENT
We plan and intend to manage and perform all data analysis at UDSM. The investigator
under supervision will carry out the statistical data analysis. Data from the paper
questionnaire will be entered into a computer with a password. These data will consist of
basic demographic, socio-economic and clinical data. At UDSM, data will be stored in a
personal computer with inbuilt-files for back-up storage. In parallel, separate external and
mobile back-up storage will be available. During the study period, only the investigator shall
have access to the dataset. All study participants will be assigned 6-digit codes attached to the
questionnaire and clinical work-up sheets.

7.2. STATISTICAL ANALYSIS


The data will be analyzed by Excel spreadsheet software for Windows. The Chi - square or
Fishers exact test will be used to detect differences in the frequencies of categorical
characteristics between the groups. The continuous variables will be analysed by Student ttest. A p value of < 5% will indicate statistical significance.

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7. ETHICAL CONSIDERATIONS
It is herewith assumed that the majority of patients with T1D are children without legal
power to consent. In this way, therefore, the informed consent to participate into the study
will be sought from the parent or guardian of every participant. Before the participants are
asked to agree or not to agree to be recruited into the study, the information sheet written in
Swahili or local dialect best understood by the guardian of the participant in relation to the
aims, potential risks and benefits of the study. The parents or guardians of each participant
will be given enough time for them to understand the study and think about their options.
They will be told they are free to participate or not in this research and if chooses not to
participate or to withdraw at any time, there will be no complaints. If the participant chooses
for the patient to participate, she/he will be asked to sign the Informed Consent Form
administered by the interviewer. Capillary blood samples will be collected for measurement
of FBG after a finger prick.

8. DISSEMINATION OF RESEARCH FINDINGS


The research findings for this study will be disseminated through sharing of the report by the
district health team together with clinicians, pharmacist and nurse practitioners at Chamwino
District Hospital. Further, we plan to share the research findings with wider scientific
communities through presentations with School of Health Sciences (SoHS) and UDSM as
well as publications of the manuscript in a local journal.

9. LIMITATION OF THE STUDY


The proposed study is the first to be conducted in Tanzania. T1D primarily affects children
thus; most of the information will be collected from parents and guardians. In addition, a
substantial proportion of individuals in rural areas are likely to be illiterate, with poor
residence thus compounding issues of proper reporting of insulin types and labels as well as
dosages. The study will also require access to hospital data which are also likely to be
missing or incomplete.

10.

PROJECT ACTIVITIES AND MILESTONES

Table 1 Project progression


Project Activities

May

June

11

July

Aug

Sept

Submission of the protocol for UDSM


review and approval
Development of Tools and Standard
Operative Procedures
Patient sampling, recruitments and
data collection
Data cleaning, analysis and scientific
discussion
Writing, production of research report
and dissertation of findings

11.

BUDGETARY ALLOCATION AND ITS JUSTIFICATION

Table 2: Budget summary in TZS


Expense

Justification

Sum (TZS)

Supplies & Services


Equipment

1 glucometer and disposable prickers and gloves will be


needed for FBG measurements estimated at 100,000.
Directly Incurred Costs

100,000

Transport and
subsistence

Site visits, meals and accommodation for 1 month for the


investigator and 1 assistant, at a rate of 25,000 per day/
twice weekly for 1 month.
Payments for support for data entry and cleaning prior to
the analysis by the investigator
Office supplies are required, which include paper, staplers,
and cost for printing.
Dissemination

400,000

Payment for printing and production of 5 copies of the


research report @ 25,000 each.

125,000

Data management
and analysis
Printing and
stationery
Research reports
production
TOTAL

150,000
75,000

850,000

12.

REFERENCES

1.

Muze, K.C. and E.S. Majaliwa, Type 1 diabetes care updates: Tanzania. Indian J
Endocrinol Metab. 19(Suppl 1): p. S12-3.
Chandler, C., et al., Insulin temperature and stability under simulated transit
conditions. Am J Health Syst Pharm, 2008. 65(10): p. 953-63.
Pingel M, V.A., Stability of insulin preparation. Diabetes, 1972. 21: p. 805-13.
IDF, Pocketbook for management of diabetes in childhood and adolescence in underresourced countries. 2013.
Gregory, R., S. Edwards, and N.A. Yateman, Demonstration of insulin transformation
products in insulin vials by high-performance liquid chromatography. Diabetes Care,
1991. 14(1): p. 42-8.
Vimalavathini, R. and B. Gitanjali, Effect of temperature on the potency &
pharmacological action of insulin. Indian J Med Res, 2009. 130(2): p. 166-9.

2.
3.
4.
5.

6.

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7.

8.
9.
10.
11.
12.
13.

Brange, J., S. Havelund, and P. Hougaard, Chemical stability of insulin. 2. Formation


of higher molecular weight transformation products during storage of pharmaceutical
preparations. Pharm Res, 1992. 9(6): p. 727-34.
Albergo, R., et al., Insulin storage and injection recommendation. Diabetes Care,
1992. 15(8): p. 1113-4.
Brange, J., et al., Chemical stability of insulin. 1. Hydrolytic degradation during
storage of pharmaceutical preparations. Pharm Res, 1992. 9(6): p. 715-26.
Allen, S.C., A cool storage pot for insulin in rural Africa. Med J Zambia, 1982. 16(4):
p. 83-4.
Arya, S.C., Insulin storage in a clay pot. Ann Saudi Med, 2000. 20(5-6): p. 491;
author reply 491-2.
Al Shaibi, K., et al., Storing insulin in a clay pot in the desert causes no loss of
activity: A preliminary report. Ann Saudi Med, 1999. 19(6): p. 547-9.
CDC. Chamwino district council profile. [cited 2016 21 December]; Available from:
http://www.chamwinodc.go.tz/pages/council_profile.php.

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