EVALUATION OF NITRATE REDUCTASE ASSAY FOR DETECTION OF MULTI-DRUG RESISTANT MYCOBACTERIUM TUBERCULOSIS AMONG PATIENTS AT NATIONAL TUBERCULOSIS REFERENCE LABORATORY ZARIA NGERIA

ABSTRACT

Multi-drug resistant tuberculosis (MDR-TB) remains a major challenge for the control of tuberculosis worldwide. Susceptibility testing of Mycobacterium tuberculosis isolates to first line drugs is therefore necessary to ascertain the diagnosis of MDR-TB. A total of 437 re-treatment patients’ samples were screened for Acid Fast Bacilli (AFB), 72 were smear positive giving a prevalence of 16.47%. Out of 72 smears positive, 62 were culture positive, 6 were culture negative and 4 were contaminant using Lowenstein Jensen medium. Out of 62 cultures positive, 57 were found to be Mycobacterium tuberculosis complex (MTBC) using immunochromatographic test while 5 were negative which were considered to be NTM (Non-Tuberculosis Mycobacteria). In this study the susceptibility of 57 MTBC isolates to isoniazid (INH), rifampicin (RIF), streptomycin (STR) and Ethambutol (EMB) was determined by Lowenstein Jensen proportion method (LJPM) and Nitrate Reductase Assay (NRA). LJ PM detected 41(71.93%) MDR-TB while NRA detected 44(77.19%) MDR-TB isolates. The occurrence of poly resistance to anti-TB drugs using NRA and LJ PM was 12% and 14% while pan susceptible were of 11% and 14% by LJ PM and NRA respectively. The sensitivities and specificities of NRA compared to those of LJPM were observed to be 98% and 98%, 64% and 68%, 89% and 92%, 80% and 77% for RIF, INH, STR, and EMB respectively. Positive predictive values were 91%, 93%, 87% and 83% for RIF, INH, STR and EMB respectively. Negative values were 80%, 92%, 67% and 90% for RIF, INH, STR and EMB respectively. Good agreement was found in all the tests with κ values of 0.63, 0.61, 0.59, and 0.61for RIF, INH, STR, and EMB respectively. The HIV/TB co-infections and HIV/MDR-TB co-infections were reported to be 9.7% and 7% respectively. Highest MDR-TB among age groups of 21-30 and 31-40 years were detected by NRA and LJPM and higher MDR-TB and TB were observed in male. The NRA has the potential to be a useful tool for rapid detection of MDR-TB in resource-limited settings because of its higher sensitivity and specificity.

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TABLE OF CONTENTS

COVER PAGE................................................................................................................................ i

FLY LEAF..................................................................................................................................... ii

TITLE PAGE........................................................................................................... Error! Bookmark not defined.

CERTIFICATION........................................................................................................................ iv

ACKNOWLEDGEMENTS........................................................................................................... v

TABLE OF CONTENTS............................................................................................................. vii

CHAPTER ONE............................................................................................................................. 1

1.0 INTRODUCTION.................................................................................................................... 1

1.1 Background of the Study.......................................................................................................... 1

1.2 Statements of Problems............................................................................................................. 5

1.3 Justification............................................................................................................................... 6

1.4 Aim and Objectives................................................................................................................... 7

1.4.1 Aim......................................................................................................................................... 7

1.4.2      Objectives.......................................................................................................................... 7

CHAPTER TWO............................................................................................................................ 8

2.0 LITERATURE REVIEW...................................................................................................... 8

2.1 Mycobacteria............................................................................................................................ 8

2.1.1 Classification of the Genus Mycobacterium........................................................................ 8

2.1.3 Growth and Metabolic Characteristics of Mycobacteria...................................................... 10

2.1.4 Mycobacterial Antigens........................................................................................................ 11

2.1.5 The M. tuberculosis Genome................................................................................................ 11

2.1.6 The Origins and evolution of Mycobacterium tuberculosis.................................................. 12

2.1.7 Epidemiology of tuberculosis............................................................................................... 13

2.1.8. Pathogenesis and Virulence of Tuberculosis....................................................................... 15

2.1.9 Primary infection.................................................................................................................. 15

2.1.11 Pulmonary tuberculosis....................................................................................................... 17

2.1.13 Diagnosis of tuberculosis.................................................................................................... 18

2.1.13.1 Mantoux test..................................................................................................................... 18

2.1.13.2 Chest X-ray...................................................................................................................... 19

2.2 TB Drug Susceptibility Testing............................................................................................... 22

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4.3 Drug susceptibility of Mycobacterium tuberculosis complex by Nitrate Reductase Assay.... 48

4.4 Drug Susceptibility of Mycobacterium tuberculosis Complex by LJ Proportion Method...... 53

4.5 Evaluation of drug susceptibility of Mycobacterium tuberculosis using Nitrate Reductase

Assay and LJ proportion Method.................................................................................................. 53

4.6 Prevalence of MDR-TB in Relation to Age Group................................................................. 56

4.7 Prevalence of MDR-TB in Relation to Gender....................................................................... 56

4.8 MDR-TB/HIV co-infection based on Age Group.................................................................. 56

4.9 MDR-TB/HIV Co-infections based on Gender...................................................................... 56

4.10 TB/HIV Co-infections patients based on age group............................................................. 61

4.11 TB/HIV Co-infections patients based on gender.................................................................. 61

CHAPTER FIVE.......................................................................................................................... 64

5.0 DISCUSSION........................................................................................................................ 64

CHAPTER SIX............................................................................................................................. 69

6.0 CONCLUSION AND RECOMMENDATIONS.................................................................. 69

6.1CONCLUSION....................................................................................................................... 69

6.3 RECOMMENDATIONS....................................................................................................... 70

REFERENCES............................................................................................................................. 71

APPENDICES.............................................................................................................................. 92

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LIST OF FIGURES

Figure 4. 1: Prevalence of TB by Microscopy............................................................................... 49

Figure 4. 2: Percentage distribution of culture.............................................................................. 50

Figure 4. 3: Comparison of MDR-TB detection by Lowenstein Jensen Proportion Method and

Nitrate Reductase Assay based on Age group.............................................................................. 57

Figure 4. 4: Comparison of MDR-TB detection by Lowenstein Jensen Proportion Method and

Nitrate Reductase Assay based on Gender................................................................................... 58

Figure 4. 5: Prevalence of HIV among MDR-TB Patients based on Age Group......................... 59

Figure 4. 6: Prevalence of HIV among MDR-TB Patients based on Gender............................... 60

Figure 4. 7: Prevalence of HIV among TB Patients based on Age group..................................... 62

Figure 4. 8: Prevalence of HIV among TB Patients based on Gender.......................................... 63

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LIST OF TABLES

Table 4. 1: Drug Susceptibility by LJPM and NRA...................................................................... 51

Table 4. 2: Drug Susceptibility of Mycobacterium tuberculosis Complex by Nitrate Reductase

Assay............................................................................................................................................. 52

Table 4. 3: Drug susceptibility of Mycobacterium tuberculosis complex by LJ Proportion method

54

Table 4. 4: Evaluation of drug susceptibility testing of Mycobacterium tuberculosis using Nitrate

Reductase Assay and LJ proportion Method................................................................................ 55

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LIST OF APPENDECIS

Appendix I : Result of Nitrate Reductase Assay showing susceptible isolates............................ 92

Appendix II: Nitrate Reductase test result showing Resistant isolates........................................ 93

Appendix III: LJPM result for resistant isolates........................................................................... 94

Appendix IV: LJPM result for susceptible isolates....................................................................... 95

Appendix V: Preparation of Working Solution of Antibiotics..................................................... 96

Appendix  VI:  Questionnaire    ………………………………………………………………….97

Appendix VII: Inform consent form…………………………………………………………….98

Appendix VIII: Ethnical approval …………………………………………………………….103

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LIST OF ABBREVIATIONS

ACP

AFB

AIDS

ATCC

BCG

Bp

BSL

C

CC

CFU

CR

DMSO

DNA

DR

DST

EMB

G

HIV

ICT

IJTLD

INH

InhA

Acyl carrier protein

Acid-fast bacilli

Acquired Immunodeficiency Syndrome

American Type Culture Collection

Baccile Calmette Guerin

Base pair

Biosafety level

Cytosine

Critical Concentration

Colony Forming Unit

Compliment Receptors

Dimethyl sulphoxide

Deoxyribonucleic acid

Drug resistant

Drug Susceptibility Testing

Ethambutol

guanine

Human immunodeficiency virus

Immunochromatographic Test

International Journal of Tuberculosis and Lung Disease

Isoniazid

Enoyl-acyl carrier protein reductase

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IPM

IUATLD

KasA

KatG

KNO3

LJ

LJPM

LPA

MDR-TB

MGIT

MIC

MODS

MTB

MTBC

MTT

NADH

NALC

NAOH

NRA

NTBLCP

NTM

NTRL

OADC

Indirect Proportion Method

International Union against Tuberculosis and Lung Disease

β-ketoacyl-ACP synthase

Catalase-peroxidase enzyme

Potassium nitrate

Lowenstein-Jensen

Lowenstein-Jensen proportion method

Line probe assay

Multidrug resistant tuberculosis

Mycobacterium Growth Indicator Tube

Minimum inhibitory concentration

Microscopic observation drug susceptibility