WOLLOUNIVERSITY

COLLEGE OF NTURAL SCIENCE

SCHOOLE OF BIOSCIENCE AND TECHNOLOGY

DEPARTMENT OF BIOTECHNOLOGY

VIRAL LOAD AND MICROBIAL IDENTIFICATION

BY
WORKNEH MEKETE
ID: 5750/11
AN INTERSHIP REPORT SUBMITTED TO DEPARTMENT OF
BIOTECHNOLOGYWITH THE PARTIAL FULFILLMENT OF

THE COURSE INTERNSHIP

Adiviser: Name ChanieDerso(Asst.prof)

AMAHARA PUBLIC HEALTH INSTITU DESSIE BRANCH

JUNE 2014

DESSIE ETHIIOPIA
 Acknowledgement

First of all I would like to thank GOD for giving the strength and always with us. Next I would like to
thank Amhara public health institute DESSIE branch for allowing me to do experiment to gain the
practical knowledge in the field of Biotechnology. I would also like to thank department head of
biotechnology Tadesse Abate (Asst.prof) and supervisor ChanieDersso (Asst.prof) for their permission
cooperation and to all APHIDB worker's MeketeMulate, and other 7 experts who support me during the
internship, and I would like to thank ChanieDersso(Asst.prof) for his great support and advise to prepare
this report. Finally I would like to say thank my department student who done the internship with me.
 LIST OF ABBREVIATIONS

APHIDB: AMHARA public health institute DESSIE branch

BSC: Biosafety Cabinet

DST: Drug Sensitivity Test

EID: Early Infant Diagnosis

HIV: Human Immunodeficiency

HPV: Human Papilloma Virus

RTPCR: Real Time Polymer Chain Reaction

VL: Viral Load

Content Page

Acknowledgement…………………………………………………..…………………i

List of abbreviation………………………………………………………………..…..ii

Table of content…………………………………………………………………..…….iii

Abstract..............................................................................................v

1 Introduction.....................................................................................1

1.1. Geographical Profile ..................................................................2

1.1.1Geographical Location.......................................................................................2

1.1.2. Historical Background............................................................................2

1.3. 4. Core Function and activities .................................................................2

1.1.4. Mission and Vision.. ......... .........................................................3

1.1.4.2 Mission...........................................................................................................3

1.1.4.2 Vision..............................................................................................................3

1.1.5 Objective of APHIDB...................................................................3

1.1.5..1. General Objective ...........................................................................................3

1.1.5.2. Specific Objective ...........................................................................................3

1.2. Organizational Structure................................................................4

1.2.1. Major activity and focus of the department ...........................................................5

1.2.2. Main objective of the internship...............................................................................5

1.2.2.1. General objective…........................................................................................... 5

1.3. Weakness and Strength of APHIDB......................................................6

 1.3.1 Weakness.................................................................................................................6

1.3.1. Strength……..........................................................................................................6

2. Main activity and discussion …….………………………….............7

2.1 Description of assigned tasks…........................................................................7

2.1.1 Viral load detection…..............................................................................................7

2.1.2. Microbial identification….......................................................................................9

2.2 Acquired knowledge, skill and merits of the internship………….........……………...9

2.3 Correlation of the internship activity and University knowledge………………...…..10

2.4 Major difficulties challenges and solution during internship……………………...…..10

3. Conclusion and Recommendation..........................................................11

3.1. Conclusion.. ...................................................................................................................11

3.2. Recommendation...........................................................................................................11

4. Reference……………….………………......................................................12

List of figure:-
Fig 1 structure of organization…………………………………………………………………………………..4
 ABSTRACT

APHIDB is one of the research centers under AMHARA regional research institute. The institute
has a mandate to establish and maintain a high-quality and sustainable laboratory system
throughout the sub-region. It also delivers quality and accessible laboratory services related to
the occurrence, causes, prevention and diagnosis of major diseases of public health importance
and to establish and support National Laboratory Quality Assurance programs and systems. In
the institution different activities have been performed, from those our internship concern about
viral load detection and micro biological laboratory. Detection of VL is performed by a machine
called Abbott RT-PCR. This is an automated machine also used for the diagnosis of EID,HPV
and covid-19.The VL detection contains the step sample reception, extraction and purification,
master mix and detection. From those except reception the other is done by the machine. The
other concern is the identification of the presence or absence of pathogenic microbe in patient
sample. This is identified by inoculating of sample on artificial prepared media. If microbe is
grow then its drug sensitive test and appropriate antibiotics is allowed to patient.
 1, INTRODUCTION
AMHARA Public Health Institute DESSIE Branch (APHI_ DB) is one of the research centers
under the AMHARA regional research institute (ARRI). The institute has three dominant
entities; namely medical and public health laboratory, research and technology transfer, and
public health emergency management. The laboratory wing provides technical support to health
facilities found in the six zones provides and serves as an in-service training center for various
capacity-building training, referral and specialized laboratory test sites, performs public health
laboratories for epidemic prone disease and conducts an external quality assessment, laboratory
monitor ship and accreditation. The institute has a mandate to establish and maintain a high-
quality and sustainable laboratory system throughout the sub-region. It also delivers quality and
accessible laboratory services related to the occurrence, causes, prevention and diagnosis of
major diseases of public health importance and to establish and support National Laboratory
Quality Assurance programs and systems. Internships enable students to acquire skills, which
cannot be learned in the classroom environment, while employers obtain access to low-cost labor
and reduced recruitment costs. Interns develop interpersonal skills, team-working skills,
professionalism and customer management experience.
 1.1. Geographical profile

1.1.1. Geographical location

APHIDB is located in AMHARA regional state in south WOLLO DESSIE, which is the
capital city of south WOLLO 401km far from ADDIS ABABA and 490km from

1.1.2, Historical Background

The AMHARA Public Health Institute DESSIE Branch (APHI_DB) was established in 1982
as a regional laboratory research center and renovated as a branch public health institute in 2016.
The institute has three dominant entities; namely medical and public health laboratory, research
and technology transfer, and public health emergency management. The institute has a mandate
to establish and maintain a high-quality and sustainable laboratory system throughout the sub-
region. It also delivers quality and accessible laboratory services related to the occurrence,
causes, prevention and diagnosis of major diseases of public health importance and to establish
and support National Laboratory Quality Assurance programs and systems. To realize its
mission, the institute benefits from the generous support of the Ethiopian and regional
governments and various national and international organizations. Some organizations that
support the institute include the World Health Organization through its Global Fund, Centre for
Disease Control, Japan International Cooperation Agency, International Center for AIDS Care
and Treatment Programs (I-CAP) and various non-governmental organizations through joint
projects. APHIDB providing service for East AMHARA region of six zones which have a total
population of more than 7,800,000.

1.1.3 Core Function and activities

Core functions and activities of APHIDB are: molecular (viral load RT PCR, EID,HPV)
Analysis, gene expert Clinical chemistry Analysis, Hematology analysis, Immunohematology
analysis, Public health micro biology Analysis, General micro biology laboratory,, Public health
chemistry Analysis, Quality assurance. Research activity and technical support on health
laboratory serves also included on its activity.
 1.1.4 Mission and Vision

1.1.4.1 Mission

To improve public health service by generating and delivering scientific evidences,

strengthening public health emergency management and providing quality laboratory service for
best public health interventions.

1.1.4.2 Vision
To be one of leading social health problem solving institute in Ethiopia.

1.1.5. Objectives of APHIDB

1.1.5.1, General objectives

 Conduct different researches based on the basic regional public health problems as well generating
and adapting scientific technologies and disseminating results to the beneficiary those of by having
utility technologies with a view to resolving outstanding health problem.
 .Strengthening public health emergency management through identifying public health threats
coordinate preparedness prevent occurrences in advance.provide rapid response whenever it occurs,
rehabilitate and recover affected communities due to emergencies.
 .Strengthening the capacity of laboratories of all levels to ensure quality diagnostic service in the
region.
 .Enhancing good governance in public health scheme for equity.
1.1.5.2 Specific objectives
 To asses patient costumers satisfaction level on clinical laboratory service provided by APHI.
 To asses association b/n patient satisfaction with clinical laboratory service at APHI and socio
demographic characteristics of patients.
 1.2 Organizational structure
APHIDB
APHI_DB

Reception
room
Reception room
PCR detection room
Molecular biology room

Microbiological room
Money cash room

Extraction room Analytical chemistry room

Gene expert Biohazard room

Culturing room
Head office Figure 1.1 Organizational structures of APHI_DB

1.2.1. Major activity and focus of the department

1, Sample referral testing: -

Provide testing service provisions on VL, EID, Covid-19 test, HPV, RT PCR, CD-4, Gene X-
pert, Clinical micro biology(culture), public health(water bacteriological analysis), back up
testing service on VL ,EID & Covid-19 for other testing sites.

2, Capacity Building Service:-

Supporting supervision, training Provision, support on document development &customization
for other regional service, participation and implementation, reagent preparation &distribution .
 1.2.3 Main objective of the internship

1.2.3.1, General objective

 To gain the practical experience in different field s of Biotechnology such as
agricultural Biotechnology(tissue culture), medical Biotechnology(diagnostics
&pharmaceutical industries...), Industrial Biotechnology, beverage industries &etc
 To obtain experience from experts of APHIDB about molecular diagnosis, VL (to detect
the amount of viral DNA with in patient’s serum) and microbiology (identification To
express our academic knowledge practically and to increase our understanding. of
microorganism present in patient's sample).
 To increase the habit of team working.

1.3 Weakness and Strength of APHIDB

1.3.1 Weakness

 Lack of automated generator, while light is crossed.

 The expert does not attend on time.
 There is no BSC in microbiology laboratory.
 Lack of the application software for better data recording.
 The lab has no adequate training policy that include, onsite training and offsite training
schedule.

1.3.2 Strength:-

 The sample collection area and testing area are separated.

 The laboratories have access of basic equipment wire loop, Bunsen burner incubator,
balance, microscope, BSC Abbott machine, and generator.
 The waste management system is improved.
 The infectious and noninfectious wastes were separated.
 The contaminated media disposed in a manner to minimize hazards to personnel.
 The lab has specimen storage area prior and following testing.

2. MAIN ACTIVITIES AND DISCUSSION

2.1 Description of assigned tasks

The activity which practiced during the internship was HIV VL detection in molecular
laboratory and microbial identification in microbiology laboratory.
2.1.1 Viral load detection
VL detection is the detection of the amount viral RNA presents in patients’ blood sample.
It was done by an amazing machine called ABBOTT m2000sp and m1000rt. This is used
to prepare spacemen or isolate pure viral nucleic acid from other cell components. The
machine contains 96 plates well, from those the 3 well introduce are internal control and
the other 93 fill with the sample to be test .The Abbott Real time assay uses RT-PCR to
generate amplified product from the RNA genome of HIV in clinical specimens. The
sample was received from patient and labeled in reception room. The sample then
transferred to molecular lab room. The sample ordered and placed in tube rank, which
was done inside the BSC. After that all the necessary materials such as, extraction
reagents (mlysis, wash 1and 2, 2, micro particle or iron particle, elution buffer),
extraction kit added in the Abbott m2000sp specified place. Then the technician allowed
the computer to start. At that time the machine began by checking first the fulfillment of
the necessary material by its own. If some error is occur it view signs. After checked it
start the process and continue until finished the extraction. The purified nucleic acid
reminds, and then oligo nucleotide reagent, thermo stable polymerase enzyme and
activation reagent added in the correct order. The machine dispensed the reagent in to all
well which is called master mix. The final out put became ready placed on plate output
rack then transfer in to Abbott m1000rt for amplification. During the amplification
reaction on the Abbott m 2000rt the target RNA was converted into cDNA by the reverse
transcriptase activity of the thermo stable DNA polymerase. Denaturation, reaning,
extension occurred for 37 cycle. The process of amplification takes 3:35 hours. During
the detection, if the VL is less than 150, the virus is not detected. If the VL is around
1000 and above the virus is detected. The total time of VL takes 7:35 hours.
Reagents and their functions

1, Mlysis:Tris solution containing Guanidiniumthiocyanate detergent. Mlysis kill virus particle

and & washes for no more infections

2, Wash-1:-Acetate solution containing Guanidiniumthiocyanate and detergent used for washing

distracted virus particle.

3, Wash-2:-Nuclease free water (RNA free water)

4, Elution buffer:-phosphate solution with preservatives, elute pure RNA. So the final elution
was RNA.

5, Micro Particle:-it contain Iron & attract the RNA

Finally the result is reported. The sample detected by the machine indicates the increasing of
the viral genetic material which may be due to either the patient not used the drug properly or the
drug were expired.
2.1.2 Microbial Identification

Microbial identification is one of the most practicable laboratories in APHIDB. This is done by
growing of microorganisms in the growth media, Materials used in process are synthetically
prepared media powder, peri dish, autoclave, incubator, flame, pope, ship blood, etc..

General procedures

Sample came from reception room. Sample can be taken from throat, sputum, urine, stool, blood,
ear discharge, eye discharge. Then prepare media, the media preparation will be specific or
general media, depending on the coming sample. Media preparations have its own step which
also different from media to media. For example during BA media preparation blood is added
after sterilization, Because of hemolytic. After media is prepared sample is dispersed on media,
then which is introduce in to incubator. Gram negative bacteria are observed between 16-18
hours after incubation while Gram positive observed after 24 hours. Quality control (quality of
antibiotics) must be checked every seven days by well-known bacteria such as Escherichia coli,
S.aurus and pseudomonas. After incubation identify, which type of microbe is this. To identify
the type of microbe different techniques will be used, But APHIDB use commonly biochemical
test and microscope. Finally drug sensitivity of identified microbe measure and the patient
allowed getting the proper antibiotics.

2.2. Acquired knowledge, skill and merits of internship

Generally I have understood the activities of the institute, specifically I have understood about
the Abbott real time machine and how it conducts the viral load and general principle about how
pathogens identified in laboratory .I have also understood what is expected from me to
participate in the institute.
2.3 correlations of the internship activities and university knowledge
The internship was gave knowledge more about the VL by Abbott real time machine in health
science. In which the VL means the diagnosis of virus to detect the quantity of viral RNA in
copies/ml, that identify who have been used to the drug or not. Correspondingly, which is related
the course molecular biotechnology. In this course we have learnt about DNA extraction
purification and DNA replication, the application of PCR, use of DNA polymerase steps of DNA
replication etc. Equivalently the diagnosis of virus (VL) by Abbott real time machine from the
internship, Which also related the course diagnostic technology, So both of the internship and
University knowledge have been a great role for obtained knowledge on health science, such as
the diagnosis of individual patients and give the direction for the infected individuals, how to use
the drugs, get balanced diet and etc. Generally both of them have a great role to give service for
the society by using different technology. Identification of pathogen which is practiced in the
organization more related with microbiology and microbial biotechnology.

2.4 Major difficulties, challenges and solutions during internship

Miss adjustment:-during the internship the order of DNTP was missed during master mix. At
time the machine make sound. This was solved by rearranging its order. An other challenge was
shortage of coming sample, because of political in stability of the country.
3 CONCLUSIONS AND RECOMMENDATION

3.1 Conclusion

The Abbott Real time HIV assay is an in vitro reverse transcription polymerase chain reaction
assay for the quantization of HIV in whole blood spot. It is intended for use in conjunction with
clinical presentation and other laboratory markers as an indicator of disease prognosis and for
use as an aid in assessing viral response to antiretroviral treatment. This assay is not intended to
be used screening test for HIV or as a diagnostic test to confirm the presence of HIV infection.
Quantitative measurement of HIV levels in peripheral blood has greatly contributed to the
understanding of pathogenesis of HIV infection and has been shown to be essential parameters in
prognosis and management of HIV infected individuals. HIV RNA in plasma quantities by
nucleic acid amplification or signal amplification technology. Working area and instrument
platforms must be considered potential source of contamination. In microbial identification the
main thing is media preparation which contains the entire nutrient required for the growth of
microbes. The different samples should be inoculated in different media, which means the media
used for one sample is differing from other sample. All activities of the institute requires care
specially, in viral load room even leg glove wearing must. In general the internship is very
important because knowledge from observation and practice can easily remember.

3.2 Recommendation

All the expert of the organization has no any idea about the department of biotechnology. In the
future the department must be announced for all sectors which we will participate. In addition the
organization experts are not voluntary to participate we other than observation. Because each
activity require care there for they fear something is missing. This problem must be solved in the
future by strengthening the relationship between the University and organization. The university
should make relation with DESSIE tissue culture center to participate their students.
4. References

http://academicresearchjournals.org/ABJJB/Absteract/2019/jully/chanie.htm

Manual which is prepared VL detection by Abbott machine

Michael J. Neil L. Morgan,John S. Rickey, Gary Higton (2001).

Molecular.Abbott/int/en/products/infect.

Recorded notes during conducting the internship recording data.

WWW.molecular.Abbott.