DECLARATION

I ADAM SINDAGASYA L, I declare that this is my original work and that it not been presented

and will not be presented to any other University for similar or any degree award.

Signature: …………………..

Date: ……………………

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 AKNOWLEGDEMENT
Firstly I would like to thank god for giving me a good health during all this practical training

time. And I would like to appreciate those people who were engaged in planning this practical

training field may god bless them all.

I would like also to thank the school of biological science for their good management from the

beginning of the field time up to an end.

In additional to that I would like to thanks all staff members of college of natural and

Mathematical Science for their strong support.

Adding my special thanks to medical development for health (MDH) headquarters, including all

staff members of MDH laboratory Temeke who were very supportive during all the activities

conducted during our brief stay of practical training period. This Includes Mr. Emmanuel

Fabian, Mr. Kanje, Madam Mercy, Madam Theo, Mr. Ramadhani as well as Ms. Irene for being

supportive during this practical training time. So thanks a lot to them and God bless them in their

activities and health living.

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 ABSTRACT

The aim of this practical training was to practically do molecular biology and a biotechnology

quantify the viral load amount in blood plasma of different adult people who are infected with

HIV/AIDS and this is specifically done to infected parents or people, and look upon progress of

medicine given to them as they are directed by doctors in their respective health institutes. But

also we were able to do early infant diagnosis (EID) to which this is done specifically to infants

of infected parents an as children are delicate to collect blood from then we use Dry blood spot

(DBS).The materials used during this practical training were Cobas AmprePrep and Cobas

TaqMan (PCR) machines, sample tubes,SPU racks,K-tips tubes,micro tubes, pipette tips, bleach

,alcohol ,computer machines, bivalve, thermal shaker, barcodes crips, barcodes crips control,

gloves, centrifuges machines, refrigerators, pipettes, pens and notebook. The procedures used

were, receiving blood samples from different health centers and arranging them in order to which

they are afterward centrifuged ready for pipetting to which when it’s done the plasma obstructed

is stored in the freezers, to which later on planed time they are taken out of the freezers and are

centrifuged so as to make the clot settle an obtain a clear plasma to which they are again pippeted

and placed in a PCR machine which when done are result are displayed and ready to be

dispatched. But results are usually ranging from high positive to low positive to a stage that there

is little virus to be detected in a plasma. But for infants it’s usually different to which they have

positive and negative and mostly their results are negative.

But moreover detection of viral load in a blood plasma indicate that a person infected but high

number of virus indicates also that person is highly infected but as copies of virus decreases

means a person is in less danger than with a person with many but also fewer virus copies a

mother is allowed to breast feed his baby for six month time, but knowing our health status is

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important so as to reduce the risk of obtaining new infections and a greater cause to our coming

generations

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 TABLE OF CONTENTS

DECLARATION ............................................................................................................................. i
AKNOWLEGDEMENT ................................................................................................................. ii
ABSTRACT ................................................................................................................................... iii
TABLE OF CONTENTS ................................................................................................................ v
LIST OF FIGURES ....................................................................................................................... vi
LIST OF ABBREVIATIONS ....................................................................................................... vii
CHAPTER ONE ............................................................................................................................. 1
1. INTRODUCTION .................................................................................................................. 1
1.1. MANAGEMENT AND DEVELOPMENT FOR HEALTH (MDH) .................................. 1
1.2. OBJECTIVES ...................................................................................................................... 1
1.2.1 General objectives .......................................................................................................... 1
1.2.2 Specific objectives. ......................................................................................................... 2
1.3 HIV EARLY INFANT DIAGNOSIS TEST ......................................................................... 2
1.4 VIRAL LOAD TEST ............................................................................................................ 2
CHAPTER TWO ............................................................................................................................ 4
2. METHODOLODY .................................................................................................................. 4
2.1 Materials Used ................................................................................................................... 4
2.2 Procedures Used ................................................................................................................ 7
CHAPTER THREE ...................................................................................................................... 14
3. RESULTS AND DISCUSSION ........................................................................................... 14
3.1 Results and Observations................................................................................................. 14
3.2 Discussion ........................................................................................................................ 14
CHAPTER FOUR ......................................................................................................................... 16
4.1 CONCLUSION AND RECOMMENDATION .................................................................. 16
4.1.1 Conclusion .................................................................................................................... 16
4.2.2 Recommendation .......................................................................................................... 16
REFERENCES ............................................................................................................................. 17

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 LIST OF FIGURES

Figure 1 Samples received and arranged for testing ....................................................................... 7

Figure 2 Centrifuging Machine ....................................................................................................... 8
Figure 3 Biological Safe Cabinet .................................................................................................... 9
Figure 4 Sample Racks ................................................................................................................. 10
Figure 5 COBAS AmprePrep and COBAS TaqMan machines.................................................... 11
Figure 6 Sample results printed in hard copy ............................................................................... 12
Figure 7 DBS Paper ...................................................................................................................... 13
Figure 8 Results Printed out.......................................................................................................... 14

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 LIST OF ABBREVIATIONS
MDH………………………………… Management and Development for Health

RT………………………………………Repeated Test

DBS…………………………………….Dry Blood Spot

SPU……………………………………..Sample Processing Units

PCR…………………………………….Polymerase Chain Reaction

AIDS…………………………………..Acquired Immunodeficiency Syndrome

HIV…………………………………….Human immunodeficiency Virus

RNA……………………………………Ribonucleic Acid

DNA……………………………………Deoxyribonucleic Acid

HC………………………………………health centers

HIV-1 CS……………………………….HIV-1 Cassettes

HIV-1 ……………………………………High Positive Control

HEID……………………………………..HIV Early Infection Diseases

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 CHAPTER ONE
1. INTRODUCTION

1.1. MANAGEMENT AND DEVELOPMENT FOR HEALTH (MDH)

Management and Development for Health (MDH) is a non-profit public health organization that

focus primarily on public health service and research in the United Republic of Tanzania.MDH

seeks to promote collaboration among government and academic institutions, as well as the

private and non-profit sectors to advance the public health and health care interests of the people

of Tanzania. MDH works together with its partners to address the problems of tuberculosis,

malaria, HIV/AIDS, and other infectious diseases and chronic non-communicable diseases

within the country. The organization also seeks to improve nutrition and maternal, neonatal and

child health in Tanzania, while undertaking initiatives to advance public health research,

education, and services to improve the lives of Tanzanian people(Mwiru, Spiegelman et al.

2015).

MDH has been taking the lead in providing oversight for the high quality HIV/AIDS care and

treatment services in the Dar es Salaam and Kagera region. MDH is governed by a board of

directors, the majority of whom are senior Tanzanian experts in the healthcare and business

fields.

1.2. OBJECTIVES

1.2.1 General objectives

A healthy and prosperous society through the HIV care and treatment program, MDH provides

technical assistance in laboratory services to the care and treatment site staff to improve quality

and strengthen laboratory systems for a wide range of laboratory operations including HIV

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diagnostics and monitoring. Laboratory services in Dar es Salaam, which involves efforts to

upgrade existing laboratory infrastructure in hospital and clinics(Habiyambere, Ford et al. 2016).

1.2.2 Specific objectives.

 HIV/AIDS Strategic Information including Monitoring and Evaluation,

 Pharmaceuticals and Supply Chain Management

 TB/HIV Collaborative Services

 Prevention of Mother to Child Transmission (PMTCT) and HIV Early Infant Diagnosis

(HEID) services

1.3 HIV EARLY INFANT DIAGNOSIS TEST

Early diagnosis of HIV in infants provides a critical opportunity to strengthen follow-up of HIV-

exposed children, as infant are so delicate same as their blood collection method it is to which

Healthcare professionals collect dried-blood spot (DBS) specimens by spot specimen carefully

applying a few drops of freshly drawn blood from a finger stick or a collection heel stick onto

specially manufactured absorbent specimen collection (filter) paper. The blood saturates the

paper and should be air-dried for a minimum of 3 hours. One of the most important DBS uses is

screening the more than 4.2 million infants(Kiyaga, Sendagire et al. 2013)

1.4 VIRAL LOAD TEST

This is a numerical expression of the quantity of virus in a given volume. It is often expressed as

viral particles, or infectious particles per mL, but as we measure the HIV sample viral load

means the number of HIV copies in a milliliter (copies/ml) of blood. If the viral load

measurement is high, it generally indicates that HIV is present and replicating. Initial, untreated,

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and uncontrolled HIV viral loads can range as high as one million or more copies/ml(Puren,

Gerlach et al. 2010).

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 CHAPTER TWO
2. METHODOLODY
During our practical training at Temeke laboratory different materials were used and they were

as follows;

2.1 Materials Used

i. Polymerase Chain Reaction (PCR) Machines

The laboratory apparatus most commonly used to amplify segments of DNA via the polymerase

chain reaction (PCR), they were two of them a CobasAmpePrep and CobasTaqMan

machines for as a HIV test it’s an in vitro nucleic acid amplification test for the quantization of

HIV-1 RNA in human plasma that targets two highly conserved regions of the HIV-1 genome,

not subject to drug pressure. In doing so, it compensates for the possibility of mutations or

mismatches and increases the probability of detection.

ii. Sampling Process Unit (SPU) racks

There are tubes that are used to hold sampling processing unit tubes to which contain sample

mixtures in a PCR machine and also these racks are specifically in a machine meaning that pcr

machines do differ hence these racks also differ

iii. Cairo tubes (K-tubes)

These are tiny tubes that o carry extracted RNA from Taqman to Ampeprep so as to amplify the

copies so as results can be read in the computers

iv. Sampling tubes (s-tubes)

These are tubes that are special for PCR machine to which contain sample from the patients

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 v. Spinix machines

This is an electrical controlled machine to which it’s used mainly to mix sample by shaking them

so to allow proper mixing

vi. Centrifuges machines

a piece of equipment that puts an object in rotation around a fixed axis applying a potentially

strong force causes denser substances and particles to move outward or settle down, this is

mostly done so as to obtain a clear plasma

vii. Thermal shaker machine

This particularly used for DBS to which are done so as to dissolve the dried blood and raises in

heat required to form the mixtures

viii. Computers

There are used to keep records of all patients and their respective areas and also to print out

results so as to make it easier to fill them in a patient forms

ix. Pipette tips

There are 1ml tips that are used to suck up solution with the help of a pipette machine

x. Bleach

This is a solution that is used to disinfect the working places in case the plasma spills

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 xi. Alcohol

This was a solution that was used to vaporize the area that was disinfected by bleach so as as the

area can be dry

xii. Macro tubes

These were used to store plasma as they were extracted so as they can be stored in refrigerators

until a planned day that they will be worked in a PCR machine

xiii. Barcodes crips

There were used so as a machine can be able to know or recognize sample so as a machine can

work it through, but they are different from control crips to which they have different color

compare to the one that contain normal plasma for they are industrial made

xiv. Gloves

They were mainly used to cover our hands so as to avoid contamination or being contaminated

xv. Refrigerators

Are used to store samples so as they can be in a good state until a time that they will be worked

in a machine, mainly they are preservers.

xvi. Pens

They were used to mark tubes as according to sample also were used to write orders made in the

computer to which a machine will work them through and the sample that were used this is done

so as to keep record

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xvii. Note book

Were used to keep record of all samples done and their respective racks

2.2 Procedures Used

The procedures that were used during viral load and HEID were as follows

i. Receiving the samples and arranging samples in order

Samples were received around noon up to evening at the reception from different health

institutes to which they were arranged then by diving them new numbers that were written on top

of the patient form with a marker pen and this was done if it respective sample was seen but if

not a form and a sample weren’t given numbers.

Figure 1 Samples received and arranged for testing

ii. Sample centrifuging

After sample being received and arranged in number accordingly, they were placed inside a

centrifuging machine for as we receive a mixed blood and we need clear plasma so a machine

will make that blood sample settle and plasma will be up and blood cells down. This is done so

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as we can have clear plasma and extract it. And this is also done so as to make clot settle and its

done in respective rpm 3200 or 10 minutes

Figure 2 Centrifuging Machine

iii. Pipetting samples under the biological safe cabinet

After centrifuging them they were separated to micro tubes so as to store them in the freezers

and also if they are about to enter in a PCR machine the sample are placed in sample tubes (s

tubes),and as so to be clear the sample is a clear plasma with no blood cells or clot

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 Figure 3 Biological Safe Cabinet
iv. Entering the data (Order and Patient Record)

Before the sample are placed in a machine order is made for which will contain initials of
patients and the rack they’re in and this is for that each patient can have his results and avoid
confusion. And also patient records are there so as the institute can know how many patients are
using medicine effectively and if a dosage need to be changed and it’s done in a computer

v. Setting sample racks into the machines (COBAS AmprePrep and COBAS TaqMan
machines, PCR)

After ordering them the sample are placed into their racks before are placed into the machine as
shown below

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 Figure 4 Sample Racks
Then they are placed in a PCR machine that the machine works in principle in such a way that

the copies are amplified via the polymerase chain reaction (PCR), polymerase chain reaction, is

an in-vitro technique for amplification of a region of DNA whose sequence is known or which

lies between two regions of known sequence.

Before PCR, DNA of interest could only be amplified by over-expression in cells and this with

limited yield Cobas AmprePrep PCR machine, then the copies are in amplification again and

detection procedures by Cobas Taqman machine in which the samples are carried to the Cobas

TaqMan by the K-tips so as o copy out results

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 Figure 5 COBAS AmprePrep and COBAS TaqMan machines
vi. Printing out the Results and filling them in patient forms

As the machines are connected automatically with the computers hence when sample are done in

a COBAS ampliprep they are printed by printers from soft copy to hard copy, to which results

are always numbers in between high positive and low positive but also they can be less than 20

or target not detected indicating the virus copies are less than hence a patient is using medicines

effectively

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 Figure 6 Sample results printed in hard copy
vii. Dispatching of Results

After results are filled to each patient respectively they are sent back to their respective HC, and

they are usually being dispatched at the end of the week or if not soon after they are all done

being worked on.

2.2.1 PROCEDURES FOR DRY BLOOD SPOTS (DBS) IN HIV EARLY INFANT
DIAGNOSIS (HEID)

 Receiving sample from different HC to which arrange then according to number

 Preparing a working area with water, bleach and alcohol before starting cutting them
from DBS papers
 Then we start selecting the one among them that if full covered wholly with blood and
cut it by using scissors and place it in sample tubes, then we clean the scissors to which
its washed in water before they are used to cut another DBS then it disinfected into the
bleach then dried in alcohol

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 Figure 7 DBS Paper

 Then the selected DBS its mixed with Specimen pre-extraction (SPEX) liquid to which
will dissolve it
 After that the s- tubes are placed in a thermo shaker for ten minutes so as to allow it to
dissolve and make a solution
 After they are done they are accordingly arranged in the racks so as they can be placed in
a PCR machine
 But in HEID there are only two control positive and negative
 Then the sample are placed in a PCR machine to which later on they will print out results

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 CHAPTER THREE
3. RESULTS AND DISCUSSION

3.1 Results and Observations

Results are always displayed in a computers and are then printed out before they are written in
patient sheets,

Figure 8 Results Printed out

But according to results many Tanzanian citizen are using HIV medicine especially women and

children thence to them the virus copies even tested are lower compare to the one that do not use

medicine, and also results shows mostly infants aren’t born with the disease to which the

infection in them results from parents, but if not infected then infected mothers will have

uninfected children

3.2 Discussion

3.2.1 Viral Load Discussion

It is often expressed as viral particles, or infectious particles per mL, but as we measure the HIV

sample viral load means the number of HIV copies in a milliliter (copies/mL) of blood. If the

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viral load measurement is high, it generally indicates that HIV is present and replicating. Initial,

untreated, and uncontrolled HIV viral loads can range as high as one million or more copies/mL.

3.2.2 Dried Blood Spot (DBS) Discussion

Dried blood spots testing is a form of the test or bio-sampling where blood samples are blotted

and dried on filter paper. The dried samples can easily be shipped to analytical laboratory and

analyzed using various methods by obtain blood from the children under eighteen months in

sucking the blood from heel, finger and blotted into the filter paper or adsorbents paper for

testing viral load quality.

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 CHAPTER FOUR
4.1 CONCLUSION AND RECOMMENDATION

4.1.1 Conclusion
From the practical training conducted at MDH, this is so important laboratory study field

because through that it help people now to know their health and how to take the measure and

care of the disease detected. Furthermore this practical training is important also because it gives

people opportunities to get further studies to the other disease as well as to get opportunities in

the life and health living for future development of the people and generation as whole.

4.2.2 Recommendation
Recommendation based on the practical training conducted at MDH, for the government and

non-government organizations, the study of HIV should be promoted for the management and

development for health for future life.

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 REFERENCES
Habiyambere, V., et al. (2016). "Availability and use of HIV monitoring and early infant
diagnosis technologies in WHO member states in 2011–2013: analysis of annual surveys at the
facility level." PLoS medicine 13(8): e1002088.

Kiyaga, C., et al. (2013). "Uganda's new national laboratory sample transport system: a
successful model for improving access to diagnostic services for early infant HIV diagnosis and
other programs." PLoS One 8(11): e78609.

Mwiru, R. S., et al. (2015). "Nutritional status and other baseline predictors of mortality among
HIV-infected children initiating antiretroviral therapy in Tanzania." Journal of the International
Association of Providers of AIDS Care (JIAPAC) 14(2): 172-179.

Puren, A., et al. (2010). "Laboratory operations, specimen processing, and handling for viral load
testing and surveillance." Journal of Infectious Diseases 201(Supplement_1): S27-S36.
User, S. (2017). MDH. [online] Mdh-tz.org. Available at: https://www.mdh-tz.org/ [Accessed 4
Sep. 2017].

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