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113 Pages·2015·1.01 MB·English

Addis Ababa University
College of Health Sciences, School of Allied Health Sciences
Department of Medical Laboratory Sciences

Application of Home Validated Panel DNA Materials for Proficiency Testing
of WHO endorsed Molecular Assays: MTBDRplus and Xpert MTB/RIF
Assay

By : Zekarias Dagne
Advisors : Kassu Desta (MSc, Assistant Professor)
Co-advisor : Walelign Dessie (MSc)

A research Thesis submitted to the department of Medical Laboratory Sciences, School of Allied
Health Science, College of Health Sciences, School of graduate studies, Addis Ababa University.
In partial fulfillment of the requirements for the degree of Master of science in Clinical
Laboratory Sciences (CLS), Diagnostic and Public Health Microbiology Specialty Track.
June, 2016
Addis Ababa, Ethiopia
Research Thesis Submission Form

Zekarias Dagne
Name of investigator
Department of Medical Laboratory Sciences, School of
Allied Health Sciences, College of Health Sciences, Addis
Ababa University
Application of Home validated Panel DNA Materials for
Full title of the research project
Proficiency Testing of WHO endorsed Molecular Assays:
MTBDRplus and Xpert MTB/RIF Assay

Duration of the project From July,2015 - January , 2016
GenoType® MTBDRplus Line Probe Assay (LPA) DST
Study Area
testing RR laboratories in Ethiopia and selected GeneXpert
testing health facilities in Addis Ababa.
Total Cost of the project 46,501.00 ETB
Source(s) of Funding Ethiopian Public Health Institute.
Address of investigator Tel: [phone]
Cell phone: [phone]
P.o.Box: 1242
Email:[email protected]
Name of Advisor(s) Kassu Desta (MSc, Assistant. Professor)
Walelign Dessie (MSc),Lecturer

I
Addis Ababa University
College of Health Sciences, School of Allied Health Sciences
Department of Medical Laboratory Sciences
This is to certify that the Thesis prepared by Zekarias Dagne ,entitled: Application of Home
validated Panel DNA Materials for Proficiency Testing of WHO endorsed Molecular Assays:
MTBDRplus and Xpert MTB/RIF Assay submitted in partial fulfillment of the requirements
for the degree of Master of laboratory science in Diagnostic and Public Health
Microbiology track. Complies with the regulations of the University and meets the accepted
standard with respect to originality and quality.

Approved by the Examining Board
External (examiner) : Desta Kassa (PhD) Signature: ______________ Date : ______________
Internal(examiner) : Samuel Kinde (MSC) Signature: ______________ Date : ______________
Advisor : Kassu Desta (MSc, Assistant. Professor) Signature:________ Date : ______________
Co-advisor: Walelign Dessie (MSc) Signature:________ Date : ______________

II
Acknowledgment
I gratefully acknowledge Department of Medical Laboratory Science, School of Allied Health
Science, College of Health Science, Addis Ababa University that provided me the opportunity to
perform this work. I would like to express my deepest gratitude to my advisers Mr. Kassu Desta
and Mr.Walelign Dessie. I am also thankful to Mr. Abebaw Kebede and TB research team
members for their advice and contribution on my thesis write up. I am also thankful to the staffs
of all regional and hospital laboratories that are participated on this study. Finally, I would like to
acknowledge the Ethiopian Public Health Institute (EPHI) for the financial support.
III
Table of Content
List of Tables ........................................................................................................................................... VI
List of Figure ........................................................................................................................................... VII
Acronyms .............................................................................................................................................. VIII
Operational Definitions ............................................................................................................................ X
1 INTRODUCTION .................................................................................................................................1
1.1 Background ................................................................................................................................1
1.2 Statement of the problem............................................................................................................3
1.3 Significance of the study ............................................................................................................5
2 LITERATURE REVIEW ..........................................................................................................................6
3 OBJECTIVES OF THE STUDY ................................................................................................................9
3.1 General Objective ......................................................................................................................9
3.2 Specific Objective ......................................................................................................................9
4 METHOD AND MATERIAL ................................................................................................................. 10
4.1 Study setting ............................................................................................................................ 10
4.2 Study design and period ........................................................................................................... 11
4.3 Inclusion and Exclusion criteria ................................................................................................ 11
4.3.1 Inclusion Criteria .............................................................................................................. 11
4.3.2 Exclusion Criteria ............................................................................................................. 11
4.4 Variables of the study ............................................................................................................... 11
4.4.1 Dependant variable ........................................................................................................... 11
4.4.2 Independent Variables ...................................................................................................... 12
4.5 Laboratory methods for Panel Preparation ................................................................................ 12
4.5.1 Mycobacterium Tuberculosis Quality control strain source ............................................... 12
4.5.2 Sub-Culturing and purity check up of the Isolates ............................................................. 12
4.5.3 MTB spiking .................................................................................................................... 12
4.5.4 Extracted DNA panel Preparation from MTB isolate........................................................ 13
4.5.5 Quality control ................................................................................................................. 14
4.5.6 Panel validation ................................................................................................................ 14
4.5.7 Panel Composition and Documentation ............................................................................ 15
4.5.8 Transportation of PT panels .............................................................................................. 15
IV
4.5.9 Evaluation of Performance ............................................................................................... 17
4.5.10 Proficiency test report ....................................................................................................... 17
4.5.11 Data analysis and evaluation ............................................................................................. 17
4.5.12 Ethical Clearance .............................................................................................................. 17
5 RESULTS........................................................................................................................................... 19
5.1 PT Validation Performance at EPHI,NTRL .............................................................................. 19
5.2 Socio-demographic characteristics ............................................................................................ 21
5.3 Participating Laboratories......................................................................................................... 24
5.4 PT Performance using DNA Panel........................................................................................... 25
5.4.1 MTBDRplus assay ........................................................................................................... 25
5.4.2 Xpert MTB/RIF Assay ..................................................................................................... 26
5.5 PT performance using spiked sputum ....................................................................................... 28
5.6 PT performance using extracted DNA ...................................................................................... 28
5.7 Overall performance indicators by drug and laboratory ............................................................ 29
6 DISCUSSION ..................................................................................................................................... 31
7 LIMITATION OF THE STUDY .............................................................................................................. 34
8 CONCLUSION AND RECOMMENDATION........................................................................................... 35
REFERENCE .............................................................................................................................................. 36
9 ANNEX ............................................................................................................................................. 40
9.1 Survey questionnaire (English version)..................................................................................... 40
9.2 Sop for MGIT culture inoculation and incubation ..................................................................... 41
9.3 Sop for Preparation of Artificial Sputum .................................................................................. 44
9.4 Sop for Extraction, Amplification and Hybridization of DNA for HAIN Probe Assays ............. 47
9.5 LPA validation Result Technologist 1 ...................................................................................... 56
9.6 LPA validation Result Technologist 2 ...................................................................................... 57
9.7 Dispatch Form......................................................................................................................... 58
9.8 Proficiency Test results form .................................................................................................... 59
9.9 Declaration .............................................................................................................................. 60

V
List of Tables
Table 1: Proficiency Test Panel strain composition validated at EPHI,NTRL and distributed to
testing sites in 2015 ............................................................................................................ 16
Table 2: Performance Evaluation of NTRL,PT Validator and SRL Judicial Result in 2015......... 21
Table 3: Demographic characteristic of Personnel's working on TB Culture and DST Regional
Reference Laboratories in Ethiopia (N=27) from July 2015 to January 2016. ...................... 22
Table 4: Overall Performance of RRL Professionals for susceptibility testing of M.
tuberculosis.(N=13) from July 2015 to January 2016. ......................................................... 23
Table 5 : Relationship between demographic characteristic with Passed (≥ 95 %) score on MTBc
and drug susceptibility testing using LPA (N=13) from July 2015 to January 2016 ............. 24
Table 6: Proficiency performance of Participant Laboratories for MTBDRPlus and Xpert
MTB/RIF in 2015 using DNA panels. ................................................................................. 27
Table 7: Suitability performance of Spiked Sputum against Extracted DNA for panel testing of
MTBDRPlus and Xpert MTB/RIF assays in 2015 ............................................................... 29
Table 8: National TB Reference Laboratory Network DST proficiency testing Performance of
seven laboratories for Isoniazid (% values).......................................................................... 30
Table 9: National TB Reference Laboratory Network DST proficiency testing Performance of
seven laboratories for Rifampicin (% values) ...................................................................... 30

VI
List of Figure
Figure 1: National TB Culture and DST Laboratory Network in Ethiopia, which are involved in
the study. ............................................................................................................................ 10
Figure 2: Description of the 27 reaction zones on the GenoType® MTBDRplus strip ................ 19
Figure 3: Description of the rpoB GENE RIF Resistance Determining Regions. ......................... 20
Figure 4: Summary performance of all the participating laboratories (N=7) on LPA drug
susceptibility testing in percentage ...................................................................................... 25

VII
Acronyms
AFB Acid Fast Bacilli
ATCC American Type Culture Collection
CDR Case detection rate
CLSI Clinical Laboratory Standards Institute
DCS Dried Culture Spot
DNA Deoxyribonucleic acid
DOTS Directly observed Treatment, Short course chemotherapy
DST Drug susceptibility testing
EPHI Ethiopian Public Health Institute
EQA External Quality Assessment
FMoH Federal ministry of health
HBCs High TB burden countries
HIV Human Immunodeficiency Virus
INH Isoniazid
IUATLD International Union against Tuberculosis and Lung Disease
LPA Line Probe Assay
MC Microscopy Center
MDR-TB Multi drug Resistance Tuberculosis
NALC N-acetyl-L cysteine
ND Not done
VIII
NRL National Reference Laboratory
NTP National Tuberculosis control program
OSE On-Site Evaluation
PCR Polymerase chain reaction
PT Proficiency Testing
PVR Predictive value resistance
PVS Predictive value susceptible
QA Quality assurance
RMP Rifampin
RRL Regional Reference Laboratory
SERO Scientific and Ethical Review Office
SNNPR Southern nations, nationalities and peoples’ region
SOP Standard Operating Procedure
SRLN Supranational reference laboratory network
RRDR Rifampin Resistance Determining Region
TB Tuberculosis
UN United Nations
WHO World Health Organization
WT Wild type
XDR-TB Extensively drug resistance Tuberculosis.
ZN Ziehl-Neelsen
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