Isolation, Identification and Antimicrobial Sensitivity of Mycobacterium tuberculosis from Patients

97 Pages·2008·0.63 MB·English

Isolation, Identification and Antimicrobial Sensitivity of
Mycobacterium tuberculosis from Patients with
Pulmonary Infection

By

Eman Magzoub Mohammed Nour
(B.V.Sc., 2003, University of Khartoum)

Supervisor

Dr. Elhassan Mohammed Ali Saeed
Co-supervisor

Dr. Nageeb Suleiman Saeed
National Health Laboratory, Khartoum

A thesis submitted to the University of Khartoum in partial
fulfillment of the requirements for the degree of Master of
Science (M.Sc.) in Microbiology

University of Khartoum
Faculty of Veterinary Medicine
Department of Microbiology

December, 2007
DEDICATION

To the soul of my father, whom I miss very much,
To my loving mother who brought me up and raised me kindly
and graciously until now,
To my dear brothers Aljali and Modathir and lovely sisters
Samia, Nahla, Tagwa and Rasha, who stood beside me.
To my all friends.
With great love and gratitude
Eman

i
ACKNOWLEDGEMENTS
First and foremost my thanks and praise are due to almighty
Allah for giving me the health and strength to accomplish this work.
Next, I’m particularly thankful to my supervisor Dr. Elhassan
M. A. Saeed for his keen guidance, genuine help, advice and
encouragement. I’m also greatly indebted to my co-supervisor Dr.
Nageeb. S. Saeed, the Director of the National Health Laboratory,
Federal Ministry of Health, Khartoum, for providing fruitful
suggestions and facilities during the study.
I wish to express my appreciation to my previous supervisor,
Dr. Ahemed Zaki, Faculty of Veterinary Medicine, University of
Khartoum, for his help and valuable advice that set me straight
where necessary during the first period of this study. Also, I would
like to thank Dr. Mohammed E. Hamid, Faculty of Medicine, King
Khalid Uiversity, KSA, for his unlimited follow up and invaluable
direction.
Thanks are also extended to the staff members of El Shaab
Teaching Hospital, Abu-Anga Hospital and Tuberculosis Reference
Laboratory, particularly Mr. Magdi, Mr. Yassir and Miss Nuha for
their integration and help
Thanks are also extended to my brother Algali for his generous
financial support to complete this study.
I’m very grateful to my friends D. Mogahid. M. Elhasan,
Hytham M. Khair, Elham Elsayid., who made my period of study
both interesting and rewarding.
ii
Last, but not least, my deep appreciation is due to my family to
whom I’m greatly indebted and whose love inspired me at difficult
times.

iii
Table of Contents
DEDICATION ……………………………………………………………………….. i
ACKNOWLEDGEMENTS…………………………….……………………..……… ii
TABLE OF CONTENTS…………………. ………………………………………… iv
LIST OF ABBREVIATIONS………………………………………………………... vii
LIST OF TABLES ……………………..……………..…………………….............. viii
LIST OF FIGURES ………………………………………………………… ix
ABSTRACT …………………………………………………………………………. x
ARABIC ABSTRACT ………………………………………………………………. xii
INTRODUCTION……………………….…………………………….…..………… 1
CHAPTER ONE: LITERATURE REVIEW……………………………………… 3
1.1 Tuberculosis …………………………………………………………………. 3
1.1.1 History of tuberculosis……………………………………………… 3
1.1.2 Definition……………………………………………………………… 4
1.2 The Bacterium…………………………………………………………………. 4
1.2.1 Description of Mycobacterium tuberculosis complex 4
1.2.1.1 Mycobacterium tuberculosis ………………………………. 5
1.2.1.2 Mycobacterium bovis…………………………………………. 5
1.2.1.3 Mycobacterium africanum……………………………………. 6
1.2.1.4 Mycobacterium microti…………………………………. 6
1.2.1.5 Mycobacterium canetti ………………………………………… 7
1.2.1.6 Mycobacterium caprae…………………………………………... 6
1.2.2 Mycobacterium other than tuberculosis bacilli (MOTT)..……………. 7
1.2.3 Other related agents including Nocardia and related actinomycetes. 7
1.3 The disease…………………………………………………………………… 7
1.3.1 Epidemiology of tuberculosis…………………………………………. 7
1.3.1.1 Transmission ………………………………………………. 7
1.3.1.2 Distribution of tuberculosis in the World…………………... 9
1.3.1.3 Tuberculosis in Sudan………………………………………. 9
1.3.2 Pathogenesis of tuberculosis………………………………………….. 10
1.3.3 Methods of diagnosis of tuberculosis………………………………… 12
1.3.3.1 Clinical diagnosis…………………………………………… 11
1.3.3.1.1 Symptoms……………………………………….. 11
1.3.3.1.2 X-Rays………………………………………….. 12
1.3.3.1.3 Tuberculin test…………………………………. 13
1.3.3.2 Laboratory diagnosis …………………………………. 14
1.3.3.2.1 Microscopic Examination………………………. 14
1.3.3.2.2 Culture methods ………………………………. 16
1.3.3.2.3 Immunodiagnostic tests………………………… 18
1.3.3.2.4 Biochemical tests………………………………. 19
iv
1.3.3.2.5 Molecular diagnosis of tuberculosis……………. 19
1.3.4 Chemotherapy of tuberculosis……………………………………… 22
1.3.4.1 History of chemotherapy………………………………... 22
1.3.4.2 Drugs used for treatment of tuberculosis………………….. 22
1.3.4.3 Current chemotherapy of tuberculosis……………….......... 25
1.3.4.4 Multi-drug resistance tuberculosis………………………….. 27
1.3.4.5 The mechanism of drug resistance…………………………. 27
1.3.4.6 Drug susceptibility and resistance determination methods…. 29
1.3.4.6.1 Phenotypic methods……………………………. 30
1.3.4.6.2 Genotypic methods…………………….............. 33
CHAPTER TWO: MATERIALS AND METHODS…………………………….. 37
2.1 Patients ……………………………………………………………………… 37
2.2 Method of sample collection …………………………………………………. 37
2.3 Equipment, plastic and glassware ……………………………………………. 37
2.4 Asepsis and sterilization ………………………………………………………. 38
2.4.1 Flaming……………………………………………………………….. 38
2.4.2 Red heat ……………………………………………………………… 38
2.4.3 Hot air oven …………………………………………………………. 38
2.4.4 Moist Heat (autoclaving)……………………………………………… 38
2.4.5 Irradiation …………………………………………………………….. 38
2.4.6 Disinfection…………………………………………………………… 39
2.5 Ziehl -Neelsen stain……………………………………………………………. 39
2.6 Medium for isolation: Lowenstein -Jensen (L.J) Medium…………………….. 40
2.7 Culture method………………………………………………………………… 40
2.8 Biochemical testing …………………………………………………………… 42
2.8.1 Catalase test…………………………………………………………….. 42
2.8.2 Nitrate reduction test…………………………………………………… 44
2.8.3 Sensitivity to Para-Nitrobenzoic Acid (PNB)………………………….. 45
2.9 Molecular identification (PCR)……………………………………………… 45
2.10 Drug susceptibility testing…………………………………………………….. 47
CHAPTER THREE: RESULTS………………………........................................... 49
3.1 Epidemiological findings……………………………………………………… 49
3.1.1 Gender ……………………….............................................................. 49
3.1.2 Age Groups…………………………………………………………… 49
3.1.3 Occupation ………………………........................................................ 50
3.1.4 Analysis of patients’ number according to their tribes(possible origins) 52
3.1.5 Treatment status................................................................................... 53
3.2 Isolation and identification……………………………………………………. 54
3.2.1 Isolation……………………………………………………………….. 54
3.2.2 Growth rate......................................................................................... 55
3.2.3 Ziehl – Neelsen staining......................................................................... 55
2.3.4 Cultural characteristics......................................................................... 55
v
2.3.5 Biochemical testing…………………………………………………….. 55
3.2.6 Polymerase chain reaction (PCR)…................................................ 58

3.3 Drug susceptibility test…………............................................................. 59
CHAPTER FOUR: DISCUSSION…………………………………………… 61
Conclusion…….….………. ... ....….…. . ….. ….. ………………… …. . . 65
Recommendations ……………………………………………………………. 66
References ……………………………………………………………………. 67
Appendix …………………………………....................................................... 82

vi
List of abbreviations

AFB Acid fast bacilli
CFLP Cleavage fragment length polymorphism
DOTS Directly observation treatment short course
EMB Ethambutol
FMOH Federal Ministry of Health
GFP Green fluorine protein
INH Isoniazid
IUATLD International union against tuberculosis and lung disease
L.J Loweinstein -Jensen
LTBI Latent tuberculosis infection
MDR Multi-drug resistance
MGIT Mycobacteria growth indicator tube
MIC Minimum inhibitory concentration
MOTT Mycobacterium other than tuberculosis
MTC Mycobacterium tuberculosis complex
NALC N-acetyl-L-cysteine (NALC)
NTP National tuberculosis program
PNB Para-nitro benzoic acid
PPD Purified protein derivative
PTB Pulmonary tuberculosis
RFLP Restriction fragment length polymorphism
RIF Rifampicin
SSCP Single strand conformation polymorphism
STM Streptomycin
TAMA Total area under mycolic acid
TB Tubercle bacilli
TBE Tris-boric acid EDTA
ZN Ziehl-Neelsen’s stain

vii
List of Tables

Table Title Page
I Principal types of opportunist disease caused by mycobacteria 9
II Estimated numbers of tuberculosis cases and rates (per 100,000
population) by WHO in different regions, for the year
2004…………………...................................................................... 10
III Differential diagnosis of pulmonary tuberculosis………………. 13
IV Biochemical characteristics for distinguishing between species of
the genus Mycobacterium …………………………………… 21
V Treatment categories and recommended short course drug
regimens………………………………………………………. 28
VI Distribution of patients according to age………………………. 51
VII Results of biochemical tests………………………….................. 57
VIII Results of drug resistance………………………………………..
61

viii
List of Figures

Fig. Title Page
١ Distribution of patients suspected with pulmonary tuberculosis according to 52
gender.....................................................................................................
٢ Percentage of age groups of patients suspected with pulmonary tuberculosis 53
٣ Occupation of study populations ……………………………………………. 54
٤ Number of patients according to their tribes………………………………… 55
٥ Different treatment statuses among patients of the study…………………. 55

٦ The percentage of M. tuberculosis and other organisms in AFB positive sample 56
٧ Growth of Mycobacterium tuberculosis on Lowenstein Jensen medium ……. 58
٨ Nitrate reduction test for Mycobacterium tuberculosis complex organisms…… 59
٩ Example of PCR result…………………………………………………………. 60
10 Resistance of MTC isolates to tuberculosis drugs……………………………… 62

ix