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

  • 0 Review(s)

Product Category: Projects

Product Code: 00001432

No of Pages: 116

No of Chapters: 6

File Format: Microsoft Word

Price :

$20

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.

 

 

 

 

 

 

 

 

 

 

 

 

vi


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

 

vii


2.2.1

General considerations .........................................................................................................

22

2.2.2

Conventional susceptibility tests..........................................................................................

23

2.2.3

New rapid susceptibility tests ..............................................................................................

25

2.3

Genotypic (Molecular) Methods of Detecting MDR-TB .......................................................

30

2.3.1

Genotyping methods used in TB Epidemiology ..................................................................

31

2.3.2

Molecular Assays ...............................................................................................................

32

2.4

Molecular Basis and Mechanisms of Resistance to First-line Drugs .....................................

33

2.4.1 Isoniazid ...............................................................................................................................

34

2.4.2

Rifampicin............................................................................................................................

35

2.4.3

Streptomycin ........................................................................................................................

37

2.4.4

Ethambutol ...........................................................................................................................

37

3.0 MATERIAL AND METHODS ..............................................................................................

40

3.1

Laboratory Settings for the study ............................................................................................

40

3.2

Study population .....................................................................................................................

40

3.4

Consent of Patients and Ethical Approval ..............................................................................

41

3.5

Inclusion criteria .....................................................................................................................

41

3.6

Exclusion criteria ....................................................................................................................

41

3.6

Sample collection ....................................................................................................................

42

3.7

Microscopy .............................................................................................................................

42

3.8

Media preparation ...................................................................................................................

42

3.9

Specimen digestion and decontamination ...............................................................................

43

3.10 Isolation.................................................................................................................................

43

3.11 Identification of Mycobacterial Isolates ...............................................................................

44

3.13 Preparation of Lowenstein- Jensen Medium with KNO3 and Drugs ....................................

44

3.14 Drug Susceptibility Testing by Proportion method ..............................................................

45

3.15 Drug Susceptibility Testing by Nitrate Reductase Assay .....................................................

46

CHAPTER FOUR .........................................................................................................................

48

4.0 RESULTS ...............................................................................................................................

48

4.1

Prevalence of TB by Microscopy ...........................................................................................

48

4.2

Culture.....................................................................................................................................

48

4.2

Drug susceptibility by LJ PM and NRA .................................................................................

48

 

viii


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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ix


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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

x


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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

xi


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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

xii


 

 

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


 

 

 

xiii


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

 


Click “DOWNLOAD NOW” below to get the complete Projects

FOR QUICK HELP CHAT WITH US NOW!

+(234) 0814 780 1594

Buyers has the right to create dispute within seven (7) days of purchase for 100% refund request when you experience issue with the file received. 

Dispute can only be created when you receive a corrupt file, a wrong file or irregularities in the table of contents and content of the file you received. 

ProjectShelve.com shall either provide the appropriate file within 48hrs or send refund excluding your bank transaction charges. Term and Conditions are applied.

Buyers are expected to confirm that the material you are paying for is available on our website ProjectShelve.com and you have selected the right material, you have also gone through the preliminary pages and it interests you before payment. DO NOT MAKE BANK PAYMENT IF YOUR TOPIC IS NOT ON THE WEBSITE.

In case of payment for a material not available on ProjectShelve.com, the management of ProjectShelve.com has the right to keep your money until you send a topic that is available on our website within 48 hours.

You cannot change topic after receiving material of the topic you ordered and paid for.

Ratings & Reviews

0.0

No Review Found.


To Review


To Comment